Animal & Veterinary

Appendix E: The Cyagra Dataset

A. Response to CVM Data Requests

The Center for Veterinary Medicine (CVM) has presented its proposed risk assessment approach at several public venues since the fall of 2002. As part of the proposed approach, we have requested that investigators engaged in cloning cattle, swine, sheep, or goats share data that they might have on the health status of these animals with the Center. The intent of the data request was to supplement the published data with unpublished data generated by the developers of these animals. We thought that data on the health status of animal clones would likely be in the hands of the private sector, which might have less impetus to publish than academic laboratories. The Center promised producers that, to the extent allowed by law, if they wished, their identities could be kept confidential by FDA, and that we would not publish the specific identity and location of the animals.

We discovered that there are very few datasets describing the health of animal clones. In general, clones are monitored closely for the first few weeks of life (or through weaning). They are often then moved from "research/hospital" facilities to "farm-like" facilities, where they are often reared with conventional animals. Most producers kept fairly cursory veterinary records unless the animal was in distress. Further, because of technical issues associated with generating successful pregnancies, only a few clones tend to be delivered at one time or from one cell line. The result is that aggregating and analyzing data becomes difficult unless publications are planned in advance.

As many of the clone producers either have primary employment as academics, or continue to maintain academic appointments, there may be data available that have not yet been shared with CVM because of the investigators' desire not to jeopardize their ability to publish in peer reviewed journals. Because of CVM's pledge to be fully transparent in this risk assessment, we determined that all data submitted would be made public through the risk assessment. We obtained the express permission from the submitters of data for the public release of this data. "Publishing" this information in the assessment could preclude formal publication in a peer reviewed journal, as most high quality peer reviewed journals have a policy of being the site of first publication.

As our risk assessment methodology evolved, it was presented at public fora (the Pew Initiative for Agricultural Biotechnology's September 2002 Biotech in the Barnyard conference, the April 2003 American Registry of Professional Animal Scientists meeting in Maryland, and the FDA Science Forum of April 2003). Subsequent discussions between clone producers and agency staff resulted in investigators returning to the field to try to collect existing data, or, in one exceptional case, to generate de novo data on the health status of clones. Without exception, every clone producer or investigator contacted was willing to answer questions on aspects of clone production, gestation, delivery, and care. Many have provided data or information that we have incorporated into this risk assessment or will use in future iterations. In order to issue the risk assessment in a timely manner, however, we have had to put off our analysis of some of the datasets until the next revision of the risk assessment. We are very grateful to those producers and owners who voluntarily expended significant time, effort, and in some cases, capital, to provide information to us.

B. Cyagra Dataset

One clone producer, Cyagra Inc.,1 has been engaged in the production of cattle clones since 1999. In the late spring of 2003, Cyagra submitted an extensive database to CVM for use in the animal health component of our food consumption and the animal health risk assessments. These data were made available for CVM to use in our risk assessments with no restriction, except to protect to the extent allowed by law the identity and location of the animals, and their current owners. In order to accommodate this request, CVM issued each animal in the study a unique identification number. These numbers have been employed throughout this analysis.

Cyagra has asserted that they have provided data on all of the clones that they can trace, including those that died, or were euthanized or culled. Animals were divided into three age cohorts by Cyagra: neonates (within 24 hours), 1-6 month age cohort (between 30 and 175 days of age), and 6-18 month age cohort (187-557 days of age).

The age spread among these animal cohorts reflects key stages in physiological development of cattle. For example, digestion differs significantly among different age groups: a 2-month-old calf is just starting to use its rumen, while a 6-month-old calf is a fully developed, cud-chewing ruminant. In this case, these two calves have been grouped together even though they have substantive physiological differences, because they have more in common than, for example, a neonate and a six month old calf. For the sake of accuracy, we have classified this group as 1 to 6 months old. A 6-18 month old calf is not quite old enough to be considered an adult, as it is still growing, and the younger animals in this group will still be pre-pubertal. We have therefore decided to classify this group simply as "6 to 18 months." The distribution of animals in the cohorts is found in Table E- 1: Distribution of Cattle Clones and Comparator Populations.

Comparators were approximately age-matched animals reared on the same farms or facilities as clones. The comparators were not born at the same locations, and do not represent the same distribution among breeds as the clones. Comparator animals were not clones, but were produced by either artificial insemination (AI) or natural breeding, from either primiparous (heifers) or multiparous dams, and were all delivered vaginally. Blood samples from neonatal comparator animals were taken after colostrum administration, while neonatal clones were sampled prior to receiving colostrum.

These animals do not provide a strict biological comparator that has experienced the same treatments and conditions as the clones. For example, the culturing conditions in the embryonic phase for cloned embryos could be more closely compared with those encountered by animals generated by in vitro fertilization. These comparators are not, strictly speaking, "control" animals.

Further, given the approximate age- and breed-matching, this dataset should not be evaluated in the same manner as a tightly controlled prospective "laboratory" experiment. Rather, our opinion is that this dataset should be viewed as an attempt to compare health and laboratory test values between clones and conventional animals comprising part of national dairy and beef herds. These data were not generated or collected under "Good Laboratory Practices," and we have not attempted to audit the data except insofar as we have detected errors or requested clarification(s) from Cyagra.

Table E- 1: Distribution of Cattle Clones and Comparator Populations for Blood Analyses

 Number of Animals
neonates1 to 6 months6 to 18 monthsTotals
Clones10461867
Comparators17472183

In Table E-1, 7 (of the 46) 1-6 month clones and 2 (of the 47) 1-6 month comparators were sampled in the neonatal group. The 1 to 6 month and 6 to 18 month cohort information was collected within a relatively short time frame. These data may best be thought of as a "snapshot" view of the animals during their development, rather than a longitudinal study in which the same animals are followed over some period of time. In fact, only nine animals were sampled or examined at more than one time point (at birth and weaning), and of those, seven were clones and two were controls (Clone ID# 71, 72, 73, 78, 79, 119, and 132; Control ID# 135 and 162).

1. Description of Clones

All clones were derived from actively dividing cells from skin biopsies; recipient oöcytes were obtained from commercial abattoirs. After 7 or 8 days of in vitro culture, morula or blastocyst stage reconstructed embryos were implanted into recipient Holstein heifers. Pregnancies were monitored closely, and with few exceptions, clones were delivered via Caesarean section (C-section) to reduce the risk associated with birth. Blood samples were drawn from the neonates prior to colostrum administration.

Table E-2 summarizes the information on samples taken from calves within the first 24 hours of birth. Some of the animals in the Cyagra dataset required some supportive care immediately after birth (e.g., glucose, warming, or supplemental oxygen), and many (n=29 out 134) received umbilical surgery after birth. Enlarged umbilical vessels which do not close naturally after birth are an identified hazard for clone calves, and many of these calves received surgery to prevent complications such as umbilical infections and bleeding (see subsequent discussion on veterinary examinations and health status). This appears to be a fairly common problem in clones, and may be associated with poor placentation. However, no direct causal attribution can be made at this time to any particular developmental pathway causing the umbilical problems.

Health anomalies noted in surviving animals for which there are no additional follow-up data include diarrhea, fever, anemia, heart murmur, and slight contracture of the flexor tendons (referred to as "contracture").

Of the 134 clones in this review, 28 were stillborn, died, or were euthanized within 48 hours of birth, leaving 106 animals (or 79 percent) alive two days after birth. At the time that data were collected on these animals (late March 2003), 67 were alive (64 percent of those surviving to 48 hours, or 50 percent of those born or delivered). Eleven (10 percent) of the animals alive at 48 hours died within approximately one and a half years later. These data are summarized in Table E-2. Of the eleven deaths between 48 hours and one and a half years later, Cyagra considers two deaths not related to cloning, and the other nine as "related, possibly related, or questionably related" to cloning. Of those fitting the "related (to some degree) to cloning" category, one was clearly a fetal developmental anomaly: flexor tendon contracture ("contracture"); three experienced difficulties with the umbilicus ultimately leading to death either via infection or adhesions; two had gastrointestinal problems with bloat or adhesions; two had circulatory problems; and one animal was euthanized for "failure to thrive."

Table E-2: Summary of Outcomes for Clones Not Surviving Birth

Animal NumberBirth Weight (kg)Age at Death (days)Problems NotedCause of Death
3NP10Abnormal deliveryStillborn
6NP16NPAccident; hung in stall
11NP0Abnormal deliveryEuthanized
12NP0Ruptured uterus in recipientStillborn
13NP0NPUnknown
1433.213Contracture2, umbilical infectionSepticemia
1650.02Slack abdomen, umbilical problems, breathing difficultiesFailure to transition to neonatal circulation
1868.20Polycystic kidneysStillborn
1969.10Umbilical problems, flaccid abdomenStillborn
20NP0Abnormal developmentEuthanized
2345.50Abnormal development, internal bleeding, umbilical problemsEuthanized
28NP0NPStillborn (C-section)
29NP0Abnormal developmentEuthanized
3176.80Abnormal renal developmentEuthanized
34NP0NPStillborn (C-section)
43NP1DiarrheaRotavirus
47NP0NPStillborn (C-section)
4854.50NPStillborn (C-section)
49NP0NPStillborn (C-section)
51NP0Flaccid abdomen, "bulldog"Stillborn (C-section)
52NP0NPStillborn (Fetotomy)
5459.10Reverted to fetal circulation, cardiac, neurological problemsEuthanized
57NP23Ruptured abomasumRuptured abomasum
63NP60Loss of hair, appetite, muscleEuthanized/failure to thrive
6561.43LethargicGI transit; adhesions from umbilical bleeding
6654.6149Contracted tendons, recurring bloat, large umbilicus requiring surgeryBloat/GI motility problems
68NP0 (2 weeks premature)PericarditisUnable to determine
77NP47Umbilical problemsSevere contracture, unresponsive to therapy
80NP1DiarrheaRotavirus
86NP0Severe contracture, fluid filled bellyEuthanized.
92NP0Depressed, pus in umbilicusUnable to determine
95NP0Severe contractureEuthanized
97NP0Severe contracture, fluid filled bellyEuthanized
10545.50Severe twisting of neck, contractureEuthanized
107NP2Hypoxemia, rapid deteriorationEuthanized
109NP0Abnormal developmentEuthanized
113NP22NephritisPyelonephritis3/ umbilical infection
123NP9Contracted front fetlocksPyelonephritis/ umbilical infection
125NP0Severe contracture, rotationEuthanized

1NP = Not provided

2Contracture is a condition in which muscles have a fixed, high resistance to stretching due to fibrosis of the tissues supporting the muscles or joints, or from disorders of the muscle fibers.

3Pyelonephritis is an inflammation of the kidney due to bacterial infection.

2. Evaluations Performed

Several types of information including veterinary records, clinical chemistry measurements, hemograms,2 and urinalysis are provided in this dataset. Not every collectable data point has been provided for each animal. Some information is unavailable because use of the data in a review such as this was not anticipated at the time the data were collected. In addition, dispersal of clones to their ultimate owners limited data collection to the degree to which owners made information or animals available. Nonetheless, this is the largest collection of information on the health status of non-transgenic clones of which we are aware, and the most detailed with respect to health status and laboratory tests.

The dataset includes information on the following:

  • Breed from which donor cells were collected
  • Gender of the donor
  • Birth date of the clone
  • Birth status (alive, stillborn)
  • Birth weight
  • Perinatal health status and veterinary/supportive care provided
  • Health status of animals between two and twelve months of age
  • Veterinary care, including treatment with drugs, surgery, or other therapeutic interventions
  • Standard blood chemistry assays (Large Animal Panels)
  • Assays for serum Insulin-like Growth Factor-1 (IGF-1), estradiol-17ß, amylase, cholesterol, and bile acids
  • Complete blood counts (CBC) and differentials
  • Standard urinalysis

Comprehensive veterinary examinations were performed by licensed cattle veterinarians. Blood samples were drawn within a few hours of birth, or at the time of veterinary examination. For CBC, blood was collected into standard EDTA-treated collection tubes; additionally, two unstained and unfixed air-dried smears were provided. For chemical analyses, whole blood was collected, allowed to clot, and the serum fraction separated by centrifugation. Laboratory analyses were all performed at the Cornell University's Animal Health Diagnostic Laboratory.

3. CVM's Analysis of Cyagra Data: Method

Our goal in evaluating the Cyagra dataset has been to determine whether extensive interrogation of the health status of the clones, including clinical chemistry and hematology, could

  1. Distinguish clones from comparators;
  2. Determine whether the health status of the clones was inferior to conventional animals and offer a predictor of a successful outcome; and
  3. Determine whether any of the information indicated concerns regarding animal health or food safety.

We note that this was not a "blinded" analysis of the provided data. No attempt was made to disguise the identity of the animals, and whether they were clones or comparators. CVM personnel engaged in performing the evaluation included veterinarians, animal scientists, toxicologists, and risk assessors, with extensive training in evaluating clinical and physiological measurements of animals traditionally consumed as food in the US.

For the overall health status of animals, the veterinary records were reviewed for notations indicating therapeutic interventions (including administration of colostrum, vaccines, dehorning, surgeries, drug therapies, etc.). Clinical and hematologic data were compared to both reference ranges provided by the testing laboratory and to the comparator animals. Additionally, laboratory values from the comparator animals were also compared to the testing laboratory to determine the degree to which the comparator group was represented by the testing laboratory's reference range (see Results). In general, urinalysis data were only used qualitatively as confirmation of outcomes noted in the clinical chemistry (e.g., glucose, BUN or creatinine levels). Table E-3 provides a summary of the analyses performed and tabulated in the Charts indicated.

Outcomes were reviewed on an analyte basis across a cohort of animals (analyte evaluation), and on a per animal basis across analytes (animal evaluation). The questions asked for each animal and analyte tested were "How many of the total animals tested exhibited values outside the comparator/testing laboratory reference range for Analyte X?" and "How many values outside the comparator/testing laboratory reference range does Animal Y exhibit?"

Table E-3. Summary of Charts Describing Comparisons

 Clones: Reference RangeClones: ComparatorsComparators: Reference Range
Clinical Chemistry:
6 to 18 months
1 to 6 months
neonates
Chart 300
Chart 200
Chart 100
Chart 301
Chart 201
Chart 101
Chart 302
Chart 202
Chart 102
Hematology:
6 to 18 months
1 to 6 months
neonates
Chart 310
Chart 210
Chart 110
Chart 311
Chart 211
Chart 111
Chart 312
Chart 212
Chart 112

The Charts are a graphical summary of CVM's analyses. For each chart, the unique identification number associated with each animal ("ID#" or "animal number") is listed in columns horizontally across the top of the table; the analysis performed is listed in rows vertically down the side of the table. If the value being evaluated fit within the comparison range being used for that interrogation, a black rectangle was recorded in the cell corresponding to the animal column/analyte row pair ( ). If the value was outside the comparator range, but judged to be not clinically relevant, a gray rectangle ( ) was recorded. If the value recorded was above or below the clinically relevant range, an arrow indicating whether the value was greater or less than the range was inserted (??). Values that were considered to be so far out of range as to be physiologically incompatible with a healthy animal but unsupported by related clinical measurements were deemed artifact and labeled "X." For example, a calf with a blood glucose level of 4 mg/dl would be comatose or dead. If the sample came from an animal that was not comatose or in distress, and there were no other related clinical measurements normally associated with abnormal blood glucose, we assumed that the measurement was an artifact. Missing values were represented by an asterisk (*).

4. CVM's Analysis of Cyagra Data: Results

a. Comprehensive Veterinary Examinations

Comprehensive Veterinary examinations were performed on 53 clones and 2 non-clones, and included explicit evaluations of the following:

  • Demeanor
  • Gastrointestinal system
  • Posture
  • Genitals
  • Gait
  • Neurological examination
  • Body Condition
  • Peripheral lymph nodes
  • Skin and coat
  • Responsiveness of pupils to light
  • Vocalization
  • Corneas and eyelids
  • Lungs (Auscultation)
  • Umbilicus
  • Nerves
  • Weight
  • Integument
  • Heart Rate
  • Musculo-skeletal system
  • Respiration rate
  • Cardiovascular system
  • Temperature
  • Oral/Pharyngeal region
  • Feces
  • Urine

The calves in this study were examined by veterinarians specializing in cattle at roughly 1-6 months of age or at 6-18 months of age. The most consistent abnormality reported for clones was umbilical surgery, often described as umbilical hernia surgery. In some instances, the records stated, "umbilicus – had surgery." Some other comments on the umbilicus were: "had umbilical hernia surgery," "ventral hernia," and "1 ½" hernia," "fluid filled mass," "umb. stump." In the initial submission of 58 animals, 26 animals had umbilical surgery. Other abnormalities reported included two clones with musculo-skeletal abnormalities, one with slight precocious (early) mammary development, two with harsh lung sounds, three cryptorchid (undescended testicles) bull calves, and one with premature ventricular contractions (PVCs, a form of cardiac arrhythmia) every 5 – 10 heartbeats.

The two clones with musculo-skeletal abnormalities included a Holstein heifer (ID# 79) with thick withers, enlarged left carpus, and leg that deviated laterally, and an Angus heifer that was a dwarf tending to gastro-intestinal bloat (Clone #108). These are obvious abnormalities and the animals were culled. The calf with slight mammary development was a 4½ month old Jersey (Clone #87). This age is young for mammary development but the phenomenon sometimes occurs in conventional heifers if they are overfed. There is no notation of follow up to determine if the calf continued to develop precociously.

The two clones with harsh lung sounds were a Holstein heifer (Clone # 41) and an Angus heifer (Clone #58). Both also had umbilical surgery. A note at the bottom of the Angus heifer's exam sheet stated that the heifer "may not return home due to permanent lung damage." There is no indication as to whether this animal was culled. Three Holstein bull clones derived from the same cell line were diagnosed with a retained testicle (cryptorchid) (Clones #128, 130, 131). Although cryptorchidism is not common in bull calves, it is thought to be heritable and is seen with some regularity. Bulls exhibiting cryptorchidism would fail their breeding soundness exams, and would not be used for breeding,3 but would not be refused by an inspector at slaughter.

A Holstein bull calf clone (Clone #126) was diagnosed with premature ventricular contractions from a single exam, but no subsequent follow up is provided to determine whether the animal outgrew the condition or whether the animal was culled. The frequency of cardiac arrhythmias in conventional calves is unknown. Thoracic auscultation (listening to the chest with a stethoscope) or more elaborate procedures are needed to detect cardiac arrhythmias. Calves are rarely examined with thoracic auscultation unless they show signs of illness.

b. Conclusions from Veterinary Examinations

The adverse physical exam findings noted in this limited sample of clones do not present a food safety issue for several reasons. One of the precepts of this risk assessment is that animals found to have a disease or condition that would render them adulterated (e.g., unfit for food, unhealthful, unwholesome) are excluded from the food supply, as normally happens with conventional animals. Dwarf animals from conventional breeding would likely be culled depending on the extent of the physical abnormality. Pre-pubescent mammary development, lung sounds, cryptorchidism, and cardiac arrhythmias are not conditions that typically exclude animals from food use. If the disease process had progressed to an extent sufficiently severe to cause systemic changes (e.g., liver congestion, enlarged heart, edematous lungs), the carcass would be condemned on inspection at the slaughtering plant. In fact, all of these conditions occur in conventional animals.

With respect to animal safety, these conditions may pose some cause for concern. Our review of these data indicates that the clone cohort appears to exhibit a higher incidence of abnormalities than might be expected in a random sample of conventional calves. There is, however, an absence of data on the prevalence of these outcomes in contemporary cattle. As some of these defects (e.g., dwarfism, cryptorchidism) likely have a hereditary component, in the absence of information on the donor cattle and their individual histories, we cannot determine whether the defects result from the cloning process, the selection of the donor nucleus, or some combination of those factors. The clustering of cryptorchidism in clones from one cell line, for example, implies that heredity may indeed be a contributing factor in the appearance of that outcome. Comparison with datasets on animal health from other clone producers would be instructive in determining whether these health problems are common among clones generated by different methods and multiple cell lines.

c. Laboratory Values: Selection of Most Appropriate Comparator

Two comparators were available for evaluating the Clones: the Cornell Animal Health Diagnostic Laboratory ("Reference Range") and approximately age-matched, and breed-distributed cohort of animals contemporarily reared at the same farms as the clones ("Comparator Population" or "Comparators"). The Reference Range population from the Cornell Laboratory is described as follows:

"We establish reference intervals by collecting blood from at least 50 adult healthy animals. These healthy animals are obtained from a variety of sources (e.g., student- or faculty-owned). Therefore, our reference intervals are only applicable for adult animals and not young animals. Results from young animals may fall outside our reference intervals because of age-dependent changes in their analytes. For example, phosphate concentrations and alkaline phosphatase activity are higher in young animals and decrease to within reference intervals at about one year of age."
(
http://www.diaglab.vet.cornell.edu/clinpath/reference/disclaimer icon)

Follow-up conversation with the laboratory indicates that the animals used to establish the laboratory's reference range were exclusively dairy cows, and thus do not represent the beef breeds that are included in the Cyagra clone cohort or comparator cohorts, and may not include bulls. In addition, it is important to remember that the reference range is selected as a statistical distribution containing about 95 percent of the normal samples. As a result, as many as 5 percent of the test values will likely fall outside that range. Statistically, when numerous tests are run on the same animal, the chance of obtaining one or more results outside the "normal range" rises based on chance alone and not a disease state.

Table E-3a: Fraction of Blood Values Within Comparison Range

AnimalsAnalysis

Clones: Cornell Reference Range

Clones: Comparator Population

Comparator Population: Cornell Reference Range

ChartFCWRChartFCWRChartFCWR
Peripubertals
(6-18 months)
Clinical Chemistry3000.753010.993020.73
Hematology3100.733110.993120.71
Juveniles
1-6 months)
Clinical Chemistry2000.622010.962020.71
Hematology2100.592110.962120.61
Neonates
(<48 hours)
Clinical Chemistry1000.311010.901020.36
Hematology1100.611110.901120.62

FWCR= Fraction contained within range of comparison, calculated by determining the number of out of range analytes of potential clinical relevance to the total number of measurements collected in each Chart.

Table E-3a provides a summary of the Charts evaluating the clinical chemistry and hematology tests performed on the Cyagra clones compared with the comparator population, and the Cornell Reference Range. In addition, the comparator population was compared to the Cornell Reference Range. First, as cautioned by the Cornell Laboratory, the Reference Range is not a good comparator for young animals. A number of the clones and comparators fall outside the Reference Range4 but the similarity to the Reference Range increases with age for both clone and comparator populations. Approximately half the animals in the older cohort were less than one year of age, however, and all clones and comparators were less than two years of age. All of the animals in the older cohorts were still growing and thus do not match the laboratory reference adult cattle population well. Clearly, then the most relevant comparison for the clone cohorts in this review is the comparator population.

d. Conclusions Regarding Clone and Comparator Population Cohorts in Aggregate

Review of the degree to which the clone cohorts have laboratory values that fit within those of the comparator population cohorts indicates the following:

  1. Even at birth, 90 percent (107 of 119 measurements) of the hematology values, and 90 percent (272 of 324 values) of the clinical chemistry values lie within the values of the comparator population (Table E-3, Charts E101 and E111). This is particularly instructive, considering that many of the clones required some assistance immediately after birth (no similar records were kept for the comparators, but we assume that no extraordinary measures were taken, and were informed that all comparators were born vaginally). Further, clones had blood samples drawn before colostrum administration, while the comparators had blood samples drawn after colostrum administration, but within 24 hours after birth. Colostrum consumption (quantity and quality) influences certain laboratory values (e.g., globulin, total protein, GGT).
  2. The 1 to 6 month age cohorts are even more similar to each other than the neonatal cohorts: both the clinical chemistry and hematology values have 96 percent and 95 percent concordance respectively (707 of 742 of the hematology measurements and 1,404 of 1,462 of the clinical chemistry measurements for clones are within the clinically relevant ranges) (Charts 201 and 211).
  3. The 6 to 18 month cohorts are almost superimposable with respect to laboratory values (Charts 301 and 311). Only three of the 294 hematological values and seven of the 592 clinical chemistry measurements were outside the clinically relevant ranges, significantly less than would be expected by chance alone.

Based on clinical chemistry and hematology values, it is not possible to distinguish between these two cohorts (clones and comparators). The superimposability of the laboratory values and the absence of any significant health observations in the clones (based on the limited number of explicit veterinary exams) leads to the conclusion that the health of these animal clones during the 6-18 month period is not inferior to that of conventional animals.

Because we have concluded that the comparator group is the appropriate basis for comparison for the clones, all subsequent discussion regarding clinical and hematological values will be considered in that context.

e. Animal and Analyte Specific Analyses

In addition to evaluating the overall status of the clone and comparator cohorts, individual animal and analyte data were reviewed to determine if more detailed evaluations could provide either confirmation of the overall health of the animals, or to serve as indicators of potential health problems that might be present in the animals that were not detected on the comprehensive veterinary examinations. For each Chart, the following two questions were asked:

  1. "For all of the clones in this age cohort, how many of the values for each analyte were out of the range established by the comparators? (i.e., looking across each row, how many arrows or grey rectangles were present?), and
  2. "For each clone in this cohort, how many of the analytes were out of the range established by the comparators?" (i.e., looking down each column of the Chart).

There are three overall issues addressed by this evaluation:

  • Whether the laboratory values of the clones were similar to those of the comparator population on an animal-by-animal level, or whether it would be possible to distinguish between the two populations based on the clinical chemistry and hematology data. A finding of similar laboratory values would provide confidence that there were no material differences in metabolic, immunologic, and hematopoetic (blood producing) functions between clones and conventional animals;
  • Whether the clones respond to the internal (growth and maturation) and normal external (stressors, disease) environments appropriately (analyte based approach); and
  • Whether the individual values can be used to predict the long-term viability of that animal or that cohort (analyte and animal approaches combined).

A description of the parameters that were evaluated and their relation to physiological status is provided in Appendix F: The Comprehensive Veterinary Examination.

Clinical chemistry and hematology responses are best evaluated in the context of the whole animal, including its age, species, breed, husbandry, geographic location, reproductive status, and the laboratory performing the analysis. Laboratory findings complement the subjective physical diagnosis of the patient by providing objective information for the process of differential diagnosis, monitoring treatment, and formulation of a prognosis (see Appendix F). "Abnormal" laboratory measurements and examinations are often defined as those values lying outside the limits of the reference range. Determining what constitutes "normal" is more complex than simply comparing an individual value to a reference range derived from a sample of a representative population.

Laboratory tests are designed to support a clinical diagnosis based on the patient's history and clinical findings, with all of the information contributing to the final decision. Comparing clinically normal clone and comparator populations only on laboratory measurements, in the absence of disease or injury, is, at best, an exercise in attempting to identify subtle hazards that may be hypothesized to exist in animal clones. The statistical or biological significance of any value that may lie outside the comparison ranges available for this review in the absence of corroborating information on the health status of the individual animals or cohorts would be difficult to justify on scientific grounds.

Further, it is important to remember that the evaluation is only as good as the sample provided. One of the terms used to group the inconsistent outcomes that result from sample mishandling or processing errors is "artifact." Erroneous conclusions can result if the artifact is accepted as a true sample result. An artifact is suspected when the laboratory data are inconsistent with the clinical assessment or there is an inappropriate relationship between tests. For example, low blood glucose values can indicate hypoglycemia, with the accompanying clinical signs of lethargy or seizure. However, low blood glucose values in a blood test can also result from not separating the red cells from the serum in a timely fashion after drawing blood. In that case, the glucose level will be artifactually reduced. Other artifacts that can influence blood variables are lipemia (the presence of excess fats and fatty acids in the blood), hemolysis (the breakage of blood cells releasing their contents into the sample), poor collection technique, and storage for too long a time at an inappropriate temperature.

Therefore, a single value outside of a "normal range," or even a number of values outside a "normal range" in one animal does not necessarily mean (or even imply) that an animal's health is at risk. Rather, these values should serve as signals for the further investigation by experienced veterinarians and animal scientists who exercise professional judgment regarding that value in the context of weighing available evidence.

In the Analyte Evaluation, similarities and differences in analytes across animals and within age-matched cohorts were considered. One or two values outside the range could be considered normal and acceptable variation in the absence of other physiologically based evidence. Because this review attempts to use clinical values to identify potential hazards, even those few values outside the range were examined in the context of other physiological characteristics to determine if those values implied some anomalous trend. If more than two variables were out of the comparator range, we returned to the animal's entire record in an attempt to understand those values in the context of the animal, and to attempt to determine if they represented a concern for the health of the animal and therefore the safety of edible products derived from it.

In the Animal Evaluation, similarities and differences within individual animals were evaluated. For each animal, we first determined the total number of analytes that were considered sufficiently out of range to imply clinical relevance, thereby triggering further scrutiny. Those analytes were evaluated for internal consistency with other values to rule out artifacts, and then considered within the context of the animal's health records for clinical consistency between the lab work and the clinical picture (based on available veterinary exam records) of the animal.

In the following sections, we have divided our analyses into the three age cohorts: within 24 hours of birth, 1-6 months, and 6-18 months. Results that are outside the comparator range are presented on an analyte and animal-specific basis. The exception to this format is the first sub-section that discusses growth-related phenomena that span age groups.

ii. Growth-Related Phenomena

Because young animals are growing rapidly, measures of bone growth such as calcium, phosphate, and alkaline phosphatase might be expected to be elevated relative to adults. (Alkaline phosphatase is an enzyme that reflects a number of physiological parameters, and in young animals represents the activity of bone growth and development). This is, in fact, observed in the clones and comparators. Review of Charts E300 and E302 (6 to 18 months old), E200 and E202 (1 to 6 months old), and E100 and E102 (within 24 hours of birth) clearly indicates that all of the alkaline phosphatase levels, and a high proportion of calcium and phosphate levels, are elevated in both cohorts relative to the Cornell Reference range. Review of Charts E301, E201, and E101, however, reveals that clone alkaline phosphatase values are almost entirely within the range of the comparators (0/18 for 6 to 18 months, 8/46 for 1 to 6months, and 0/10 (within 24 hours of birth) values out of range). For those animals in the one to 6 month cohort, the increased levels of alkaline phosphatase occurred in the youngest animals, a finding consistent with higher rates of growth in younger animals relative to older animals.

Total protein, globulin, and albumin reflect, among other things, the immune status of the organism, which varies with age. Immediately after birth, globulin levels, which are largely comprised of immunoglobulins, are derived almost entirely from colostrum (the antibody rich first "milk" secreted by mammals). "Passive immunity" is conferred by the ingestion and intestinal absorption of immunoglobulin-rich maternal colostrum. In the two to four months after birth, a calf's own immune system begins to ramp up its production of immunoglobulins, as the circulating supply of maternally-derived immunoglobulins in milk wanes. This phenomenon can be observed in Charts E200 and E202 (Clones: Reference Range (1 to 6 months), and Comparator Population: Reference Range). Clone and comparator calf globulin values are low relative to the Cornell lab reference range because that reference range is derived from adult animals. The clone and comparator calves have not fully started to produce antibodies from their own B-lymphocytes. Review of Chart E201 (Comparison of Clones to Comparator Population), however, indicates that there were few differences between the clone and comparator populations, reflecting the appropriate age-related lag between the decrease in passive acquired immunity and endogenous immunoglobulin production.

The globulin levels that are different between clones and comparators also reflect this age-related physiological phenomenon. Clones #72 and 73 were among the youngest in the 1 to 6 month old group, and thus would be expected to have lower globulin levels. For example, comparison of the globulin value for clone #100 (174 days of age, globulin of 4.6g/dL) with clone #72 (48 days of age and globulin level of 1.6 g/dL) clearly demonstrates the age-related changes in the analyte, and appropriately reflects the normal developmental increase in endogenous globulin production.

In summary, clones and comparators exhibited entirely appropriate developmental stage responses in those laboratory values that reflect age-specific alterations. At least with respect to this dataset, clones respond appropriately to the internal signals guiding normal development and maturation.

f. Animals with Measurements at Different Developmental Nodes

There is a small sub-cohort of seven clones for which laboratory values are available for both the earliest (neonatal) developmental group and early in the second developmental node group (1-6 months). The seven calves are 71, 72, 73, 78, 79, 119, and 132.

More variability was observed among the clinical chemistry values generated from six of seven of these clones relative to the comparators than in the hematology values (for one clone, #119, no differences were noted relative to the comparators). Compare Charts E101 vs. E201 for chemistry and Charts E111 vs. E211 for hematology. In particular, gamma glutamyl transferase (GGT) values appeared lower for four of the seven neonatal clones. This observation is entirely consistent with the difference in treatment that the animals received with respect to the timing of the blood draws. For clones, blood samples were drawn prior to the administration of colostrum, while the comparators had blood samples drawn following its administration. As colostrum has been shown to have high intrinsic GGT activity, the difference between the two groups could well be due to the reflection of absorbed colostrum by the comparators (Meyer and Harvey 2004). GGT values normalized by the second time of measurement for three of the clones. Calf #73 continued to demonstrate slightly lower GGT activity (4U/L vs. comparator range of 5 - 32 U/L) at Day 48, but this value is unlikely to have clinical significance. We would have expected the globulin level to be lower also, because of the young age of this clone. There was one comparator calf with a low enough globulin level (1.3 g/dl) to allow all of the clone calf values to fall within the range, had it been included in establishing the range.

Six of the seven clones exhibited lower aspartate amino transferase (AST) values at birth relative to the comparators, but these values normalized by the time of the second blood draw (Chart E101 for neonates v. E201 for 1-6 months). Five of the seven clones had low bile acid or cholesterol levels at birth. All three of these analytes are produced by the liver, and their relatively low values may be a reflection of the changeover from fetal hepatic circulation (which bypasses much of the liver) to neonatal circulation in which the liver becomes more fully perfused. Bile acids normalized by the second measurement. The initial, relatively low creatine kinase (CK) (Clone #72), total iron binding capacity (TIBC) (Clone #73), and elevated iron (Clone #132) values resolved by the time of the second blood draw. The cholesterol level for clone #79 was low at birth, but was elevated relative to comparators in the 1-6 month blood sample.

A few measurements appeared elevated in five of the clones at the time of the second measurement. Glucose, alkaline phosphatase, phosphorus, creatinine, and the A/G ratios were elevated relative to the comparators, and anion gap, globulin and total protein were decreased relative to the comparators. These variations from the comparator range are discussed more fully in the Animal and Analyte Review portions of this Appendix.

For hematological data, complete blood count information is only available for four of the seven neonatal clones (Chart E111). Of those, only Calf #78 exhibited any values outside the range of the comparators. At birth, that animal exhibited decreased lymphocytes and decreased platelets, which normalized by the time of the second blood draw. The second blood sample (Chart E211) showed increased banded (immature) neutrophils, whose only biological significance here is that it demonstrates that the clone's bone marrow can produce normal immature neutrophils. The stimulus for the release of the banded neutrophils in this case is not known. No neonatal hematological values were available for clone #78. At the time of the second blood draw (Day 84) clone #79 exhibited decreased total protein as measured by refractometer (TP-ref), increased mean platelet volume (MPV) and decreased red cell distribution width (RDW) relative to the comparators. These variations (increase in bands for clone #78 and decreased total protein (TP) and RDW and increased MPV) are discussed in the animal and analyte review sections. Clone #79 was eventually culled (see discussion in Animal and Analyte Section).

In summary, with the exception of one clone (Clone #73, GGT), none of the values measured at birth that were out of range of the comparator group persisted into the second developmental cohort. Initial laboratory measurements taken at birth differed in clones versus comparators. Some of that difference was likely attributable to the difference in timing or source of colostrum administration between the two groups. Clones #71, 72, and 73 were derived from the same cell line. As discussed in the Animal and Analyte Review sections, most of the values that were out of range at the time of the second blood draw can either be attributed to the age of the animals or do not appear to have clinical significance.

ii. Age Range within 24 hours of birth (Charts E101 and E111)

There were 10 live clones and 17 live comparators in this age cohort.

(a) Analyte Evaluation

Despite an expectation of substantial differences in laboratory measurements between clones and comparators at birth, Chart E101 shows 27 of the 33 analytes were very similar between the groups: they had either one difference or no differences. The remaining six analytes tended to be more variable between clones and comparators.

The values out of range in the four analytes related to liver function (AST, GGT, cholesterol, and bile acids (hBA)) were low relative to comparators. AST levels, in particular, were low in 9 of 10 animals. Although these parameters can indicate zinc or vitamin B6 deficiencies, (Duncan and Prasse 2003, Meyer and Harvey 2004) have reported that "reduced AST activities (below the reference range) are noted with relative frequency in dogs and rats during pre-clinical drug trials…." We are uncertain whether similar observations have been made in cattle. Colostrum possesses an intrinsic high GGT activity (Meyer and Harvey 2004) that is passively transferred to neonates. Given that the clones did not receive colostrum until after blood samples were drawn, while comparators did, lower GGT levels in clones are not unexpected.

Low cholesterol is associated with porto-systemic shunts (abnormal liver blood circulation) in young animals. Fetal circulation provides for the bypass of the bulk of the liver tissue through a vessel called the ductus venosus in a loop including the placenta, umbilical vein, and the ductus venosus. At birth, the ductus venosus closes off; the liver is then fully perfused with blood from the hepatic artery and other components of the hepatic portal circulation. Were these low cholesterol levels to continue into the next developmental age group, there might be cause for concern. Given that the cholesterol levels appear to normalize, based on information from the seven calves that were sampled at both the neonatal and weaning time periods, and review of the overall 1-6 month cohort, there is little reason to think that the lower values in these very young animals pose a health risk. The low levels at birth are more likely a reflection of the changeover from fetal to adult circulation, possibly exacerbated by the clones' unusually large umbilical vessels, which often required surgical correction (see 1-6 month cohort discussion). The lower bile acid values observed may also be related to the transition from fetal to neonatal circulation, and are not likely indicative of any disease state.

Table E-4: Summary of Laboratory Values from Subcohort of Clones with Laboratory Measurements Taken at Two Developmental Nodes

Calf ID#ParametersCharts E101/111: BirthAge at Second Blood Draw (Days)Chart E201/211: 1-6 MonthsCurrent Status
78, Holstein ?Clinical Chemistry?AST, ?GGT (pa)54?glucose, ?alk phosPresumed healthy
Hemogram?lymphocytes, ?platelets?banding
79, Holstein ?Clinical Chemistry?AST, ?hBA65?creatinine, ?globulin, ?A/G, ?alk phos, ?cholCulled
HemogramNo data?rdw, ?MPV, ?TP-ref
71, Holstein ?Clinical Chemistry?AST, ?hBA,
?GGT (pa)
48?P, ?glucose, ?alk phosPresumed healthy
Hemogramwithin rangewithin range
72, Holstein ?Clinical Chemistry?AST, ? hBA,
?GGT, ?CK
48?anion gap, ?P, ?TP, ?glob, ?A/G, ?glu, ?alk phos, ? lipemiaPresumed healthy
HemogramNo data? MPV
73, Holstein ?Clinical Chemistry?AST, ?GGT (pa), ?TIBC, ?hBA, ?cholesterol48?anion gap, ?P, ?TP, ?glob, ?A/G, ?glu, ?GGT,Presumed healthy
Hemogramwithin range? MPV
119, red Holstein ?Clinical Chemistrywithin range36within rangePresumed healthy
Hemogramwithin rangewithin range
132, Holstein ?Clinical Chemistry?AST, ?chol, ?iron, ? % saturationNot availablewithin rangePresumed healthy
HemogramNo datawithin range

pa = presumed artifact

Other abbreviations as described in Appendix F: Comprehensive Veterinary Examination

Within range = within the range of values for the comparator population

The hemograms for the neonatal clones (Chart E111) also were very similar to the comparators: 15 of the 17 analytes had either no values out of range or just one value out of the comparator range. With the exception of clone #43, which was infected with rotavirus and died at one day of age, all red blood cell analytes were within the range of the comparator group. Three clones had lymphocyte counts lower than the comparator range.

(b) Animal Evaluation

In general, clones appeared to be more fragile perinatally than comparators. Review of Table E-2 indicates that many of the animals did not survive parturition. Of those that did survive, three were infected with rotavirus, and two, clones #43 and #80, died from rotavirus-induced diarrhea.5 Clone #43 exhibited lower AST, cholesterol, GGT, and bile acids than the comparator range, and platelet counts higher than the comparator range. Clone #80 had sodium levels that slightly exceeded the comparator range (149 mg/dL, relative to the highest comparator value of 146 mg/dL), elevated iron, and relatively high percent saturation. The elevated sodium level may have been related to electrolyte disturbances that occur with diarrhea.

Calf #78, a Holstein heifer, also had WBC within comparator range at birth and developed normally through Day 54 of age. She had low lymphocytes and low platelets, but did not present clinically with infection, and survived. Low lymphocyte counts (lymphopenia) can result from severe systemic bacterial and viral infections, disruption of the lymphatic drainage (ruptured thoracic duct), or hereditary disorders in which the production of the lymphocytes is impaired. It may also be a function of suppressed immune function in calves. In the absence of additional information, we think that these data should not be overinterpreted. Of the three clones with lymphopenia, two died from infection, but the third survived, indicating that although perinatal lymphocyte count may be a useful parameter to monitor, it is not predictive of outcome.

Clones #71, 72, 73, 78, 79, 119, and 132 were discussed in aggregate in the preceding section of the subcohort with laboratory measurements taken at two time points. Clone #79 was a Holstein heifer that was culled for poor posture and gait at 54 days of age. In addition to low AST and low bile acids, she was noted to have "very thick withers, and a general build resembling a beef calf. Her left carpus was enlarged, and her left leg deviated laterally."

Clone #75 was a Holstein heifer with no follow-up data at a later age. She exhibited AST, cholesterol, GGT, and bile acid levels that were lower than the comparator range. Clone #132 was a Holstein heifer initially presented with AST and cholesterol levels lower than the comparator range and iron and TIBC above the comparator range, but that appeared to be thriving at 50 days.

(c) Conclusions for Perinatal Period: Animal Safety

In general, clones appeared to be more fragile than comparators. Three of the ten calves died or were culled. Two of these died at one day of age of rotavirus infection, and one was culled for poor posture and gait. The major classes of adverse outcomes noted for neonates included stillbirth, umbilical bleeding/abscess/management, colostrum/passive transfer problems, and euthanasia for defects (renal, circulatory, tendon contracture, placental abnormalities, cardiac, abomasal, and ascites (increased fluid in the abdomen)). These problems also occur in conventional animals, although the incidence of the adverse outcomes appears to be higher in clones. Animals with these outcomes are readily identifiable.

The laboratory values indicated a variety of anomalies in the clones. The liver values (AST, GGT, cholesterol, bile acids (hBA)) were decreased in several animals, for reasons likely related to the placental/umbilical abnormalities. GGT levels were also low relative to the comparators, likely related to blood sampling prior to colostrum intake. Although both red and white blood cell counts are more variable than in older cohorts, both systems appear to be functioning normally in response to environmental stress (rotavirus).

Based on these data, it is not possible to use either a particular analyte or analyte profile to predict whether an individual animal, or indeed the entire cohort, will develop into normal, fully functioning healthy animals. The health and laboratory data are consistent with the hypothesis that animals that look and behave normally are most likely normal with respect to laboratory values.

(d) Food Safety

Healthy clones of this age are unlikely to be used for human food, given their potential value as breeding stock. It is also highly unlikely that clones of this age group would be fed to animals except through rendering of dead clones that occurred at parturition or by accident. In any event, the laboratory values do not appear to indicate that these animals are materially different from conventional newborns, but their physical condition at birth seems to indicate that they are more fragile than comparators, and by inference, other conventional animals.

iv. Age Range 1 to 6 months (Charts E201 and E211)

There were 46 clones and 47 comparators in this age cohort.

(e) Analyte Review

Chart E201 shows glucose values were higher for clones than comparators in six of the 42 accepted measurements (four of the values are so low as to be incompatible with life (< 2mg/dL) and thus were considered artifacts). The higher values (ranging from 88-123 mg/dL) may reflect stress responses caused by handling, dietary considerations (including proximity to a meal), or real differences in glucose metabolism. In order to determine whether the hyperglycemia was transient or sustained, urinalysis results were checked for these animals. The renal threshold (the blood level at which glucose spills over into the urine) in cattle is approximately 100 mg/dl. As none of the urinalyses tested positive for glucose, it is unlikely that the hyperglycemia observed in the blood had been sustained long enough to allow spillover from the blood into the urine. Further, no mention of increased water intake or urination was noted on any of the veterinary records of these clones, which are clinical signs of sustained hyperglycemia. Therefore, the most physiologically plausible interpretation of these elevated levels is transient hyperglycemia as a short-lived response to stress.

Total Protein (TP) is an analyte that contains globulin and albumin. Two animals had TP values that were outside the comparator range (Clone #72 and clone #73 were low). Three animals (Clones #94, 102, 128) had decreased levels of SDH relative to the comparators; three others (Clones # 56, 73, 116) exhibited lower GGT levels than the comparators. Although elevated blood levels of SDH and GGT may be indicative of liver disease, the relevance of decreased levels is unclear.

Evaluation of hemograms (Chart E211) indicated that there were no anemic animals. The one value out of range of the comparator group was an elevation, and not a decrease, in RBCs. Although other red cell indices such as mean cell hemoglobin (MCHC) concentration (4/44 clones had elevated levels relative to comparators) and red cell distribution width (RDW) (3/44 clones had lower levels than comparators) were out of comparator range, they are secondary indicators of red cell status. Hemoglobin (Hb) and hematocrit are the primary indicators of red cell status, and were effectively no different from comparators. No variables were consistently out of range for white blood cell evaluations. Four of the 44 clones had elevated basophil counts; the significance of these measurements is unclear. Twelve of the 41 clones had mean platelet volume (MPV) values that were elevated. See clone #102 below for a discussion of MPV.

(f) Animal Evaluation (1 to 6 months Age Group)

Clone #41, a 141 day old Holstein heifer, exhibited no clinical chemistry and one minor hematology value out of range of the comparators. Her health records, however, stated that she had umbilical surgery and harsh lung sounds. No further information is available on this animal.

Clone #58, a 161 day old Angus heifer had a normal veterinary exam, with indication of umbilical surgery. Four clinical chemistry values were out of the comparator range: creatinine, albumin, bile acids, and the A/G ratio were all low. Potential causes of low albumin levels include decreased production from chronic liver failure, or increased loss due to nephropathy (a kidney disease in which proteins are excreted), intestinal disease (enteropathy), or loss into a body cavity such as the thorax or abdomen. Chronic liver failure is accompanied by elevated bile acid levels; clone #58's bile acids were low. There were no other analyte or health measures indicating enteropathy or nephropathy. The low albumin level was also accompanied by a high globulin level, which could well be attributed to the umbilical abscess that was surgically removed, as the globulin response was appropriate to antigenic stimulation. The relevance of low creatinine is unknown. This calf also had four hemogram variables that differed from comparators. Hemoglobin (Hb), TP, and RDW were high, and the lymphocyte count was relatively low. The elevated Hb is consistent with the high hemolysis index of the sample, and is therefore probably not a reflection of the biology of the animal. The elevated TP was likely caused by the elevated globulin in response to antigenic stimulation discussed in the above.

Three Holstein calves (Clones #71, 72, and 73) were derived from the same cell line and were the same age at blood draw (48 days). All three presented with normal veterinary exams, elevated alkaline phosphatase levels, glucose, and phosphate levels. Clone #71 did not exhibit any other clinical values outside the comparator range. The elevated glucose measurements, as discussed above, appear to be transient and likely stress related. Clone #72, also presented with a normal veterinary exam. Her clinical chemistry measurements indicated low anion gap, TP, and globulin levels, and elevated phosphate, A/G ratio, glucose, and lipemia levels. Low anion gap is rare, and can be related to low albumin, which this animal did not exhibit. Its importance is unknown, and in this case, may simply be an anomaly. The low TP and globulin levels are likely age-related, as at this age calves are transitioning from maternal antibody to endogenous production, and there is often a lag in globulin concentration during this age. Plausible explanations for elevated phosphate, A/G, and glucose levels have been discussed previously. The lipemia level for clone #72 was 30U compared to 25U. As the lipemia index is relatively arbitrary, in the absence of corroborative health evidence, it is likely that this value has no real clinical significance. Clone #73 had low anion gap, TP, globulin, and GGT levels in addition to the elevated phosphorus, A/G, and glucose values. As previously discussed, particularly considering the very young age and high genetic merit of these animals, the elevated phosphorus and alkaline phosphatase levels are not surprising. The influence of colostrum on GGT levels has been discussed previously, although its significance in this age group is not clear. Comparison of clone #41 to clone #73 shows that the former exhibited no laboratory values out of the range of the comparators, but did have health problems, while #73 exhibited many laboratory values outside the comparator range, but no health problems.

Clone #79 was a 65-day-old Holstein heifer at the time of the blood sampling. She was culled for poor posture and gait. Clinical chemistry indicated elevated creatinine, A/G, alkaline phosphatase, cholesterol, and reduced globulin levels.

Clones #87, 88, and 89 were all derived from the same Jersey cell line, and were, respectively, 141, 140, and 131 day old heifers at the time of blood draw. Another animal derived from the same cell line died at birth from LOS-related complications. All three had umbilical surgery and were dehorned. The differences in body weight in these animals illustrate the variability seen among clones derived from the same cell line. Clone #87, the oldest at 141 days, weighed 282 lbs; clone #88 (140 days) weighed 197 lbs; and the youngest (Clone #89 at 131 days) weighed 215 lbs.

Clone #100, a 174 day old Holstein bull, had an elevated WBC (26,500 cells), along with a history of umbilical abscess that was treated surgically. The elevated WBC is an appropriate response to an umbilical abscess.

Clone #102, a135 day old Holstein heifer also had umbilical surgery, reduced platelets (241 x 103), and an elevated MPV and MCHC relative to the comparators. By itself, the latter measurement has little relevance unless anemia is present. Based on the RBC counts, this calf did not have anemia. The relatively low platelet count also does not appear to be significant; for reference, the low end of the Cornell Reference Range is 232 x103, or functionally the same number. Also, automated platelet counters may erroneously count platelets, as they tend to aggregate (clump together). The platelet smear listed platelets as adequate, corroborating that the platelet numbers were likely physiologically appropriate.

(g) Conclusion for 1 to 6 month old group: Animal Safety

The clones in this age cohort were mostly normal. Only one calf was culled for reasons of performance (poor conformation) and not animal health. Such calves are not selected for future breeding and their appearance (and subsequent culling) in a herd is not unique to clones. Culling occurs routinely in conventional breeding programs. The observation of poor conformation in a clone is interesting in that the animal providing the donor cell would likely have exhibited acceptable conformation, raising the question of whether conformation of this animal is a function of its uterine environment or changes in gene expression. Clones from the same cell line showed considerable variation in their phenotype (see clones #87, 88, and 89 above with respect to weight).

Some of the clones had overt health problems. These included the increased incidence of umbilical problems (e.g., enlargements, excessive bleeding, oomphalitis (navel infection)) tendon contracture, and cryptorchidism. Clones had umbilical extirpation (surgical removal of tissue) at a much higher rate than comparators. This increase represents a real risk to clones related to surgery. Surgical risks include complications that may arise from anesthesia and recovery from surgery, sepsis from manipulating an infected umbilicus, dehiscence (suture line not holding or infection of the suture line), and aspiration of stomach contents into the lungs. Contracted tendons also seemed to occur at a higher frequency than in conventional calves. (Tendon contracture can generally be treated successfully.) Three cases of cryptorchidism were identified. This condition is thought to be heritable, and is relatively uncommon in calves. The risk to the animal is that retained abdominal testicles can develop neoplasia (testicular cancer) in later age. The life cycle of food animals is such that bulls rarely live long enough for neoplasia to develop. In fact, the only food animals that would likely survive to develop such a condition would be breeding bulls. Given that a cryptorchid bull would fail its breeding soundness exam and would not be used for breeding stock (i.e., would be castrated and sent to slaughter when the steer reached the appropriate weight), this eventuality is not likely to occur.

(h) Conclusion for 1 to 6 month old group: Food Safety

It is not likely that clones of this age group would be consumed for food, although there may be some circumstances in which culled clones might be sent into the food supply. When the results of the laboratory analyses are considered in the context of the Cyagra clones' clinical presentation, there were no consistent analyte or physical observations indicating a food safety concern. For example, although some calcium, phosphorus, alkaline phosphatase, and glucose levels fell above the comparator range, all of the elevations can be explained by the clones' stage of life or stress level, and the increased levels observed do not represent a food consumption risk. Further, the laboratory work is consistent with clinical presentation: Calf #100 presented with both umbilical abscess and a high WBC count. In the unlikely event that this animal was sent to slaughter with a large abscess, it would be detected on inspection. The carcass would be condemned if there was evidence of systemic involvement. The abscess would otherwise be cut out and the carcass processed normally. Healthy clones of this age group do not appear to be materially different from the comparators, and would not likely pose a food consumption risk different from conventional animals.

v. Age Range: 6-18 Months (Charts E301 and E311)

There were 18 clones and 21 comparators in this age cohort.

(i) Analyte Analysis

Review of Chart 301 (Clone: Comparator Population Clinical Chemistry) indicates that there were very few differences between these two cohorts: 33 of the 33 analytes showed no or one value out of the range defined by the comparator population. Two analytes, on first impression appeared to exceed that range: estradiol-17ß (E2) and insulin-like growth factor-1 (IGF-I). On further scrutiny, these values were judged to be of no clinical relevance. Because hormones are important from a physiological and food safety standpoint, their lack of clinical significance is discussed below.

IGF-I is a hormone produced by all mammals, whose presence is necessary for growth and development. Circulating levels of IGF-I have been linked to weight gain and growth rate, and higher levels have been used as a physiological marker for superior genetics in cattle, swine, sheep, and chickens (Davis and Simmen 2000). In this study, IGF-I levels tended to be higher in male clones than in females, and in three bull calves (Clone #24, 33, and 35) were slightly increased (less than 10 percent) relative to the comparator group (respective IGF-I levels of 924, 916, and 938 ng/mL relative to the comparator range of 33-875 ng/mL).

Basal circulating levels of IGF-I vary with a range of factors, and fluctuate dramatically among individual bovines in herds (Vega et al. 1991). In an analysis of 603 conventional Angus cattle conducted 42 days after weaning, the serum concentrations of IGF-I ranged from 17 to 883 ng/mL (Davis and Simmen 2000). Basal IGF-I levels also vary between males and females, with 12 month old bulls exhibiting higher concentrations of IGF-I than steers, heifers, or ovariectomized (animals whose ovaries have been surgically removed) heifers (Plouzek and Trenkle 1991 a,b). Plasma concentrations of IGF-I are also influenced by diet composition and intake, with basal IGF-I levels significantly lower in cattle during feed restriction compared to cattle that are fed to meet maximum growth or production potential. The primary nutritional determinants of basal IGF-I levels appear to be crude protein and total metabolizable energy6 (Elsasser et al. 1989). Given that most non-transgenic clones are derived from animals of superior genetic merit for traits such as growth and development, the likelihood that their diets would be highly controlled, and the wide variability in normal IGF-I levels, the observed 10 percent elevations in IGF-I levels are of no clinical significance.

Of the five clones (# 24, 33, 35, 36, and 69) in the 6 to 18 month dataset that were identified as having plasma E2 levels above the comparators, all were bulls. These differences in E2 levels prompted closer scrutiny. The range in concentrations of these five bulls was 14.16 to 24.33 pg/mL. The range for all 18 clones in this age group was 4.28 to 24.33 pg/mL. The laboratory reference range is 10 to 40 pg/mL, while the comparator range was 4.1 to 11.41 pg/mL. As the laboratory reference range most likely included cycling females, we sought more specific information on E2 concentrations in bull plasma. We then compared the values to the Cornell Reference Range, derived from adult animals, and found that none were outside that range.

Although male mammals produce E2, little research effort has been devoted to studying the role, normal concentration, and fluctuation of endogenous estrogens in the bull. Estrogens are produced in the Sertoli cells of the testis, as well as in adipose tissue and the brain (Henney et al. 1990). Estradiol-17ß (E2) is produced when testosterone binds to cells in the hypothalamus and is converted to E2 by the aromatase enzyme. Receptors for E2 have been identified in the urogenital tracts of growing and adult male mammals of several species, and may be necessary for normal structural and functional development of the male reproductive system (Nilsson et al. 2001). The ratio of E2 to testosterone may be an important factor in male sexual behavior and libido (Henney et al. 1990).

Henney et al. (1990) attempted to relate various hormone concentrations in plasma to libido in 18 Holstein bulls aged 4 to 5 years. Mean concentration of E2 in plasma of these bulls was 10.2 pg/mL, but ranged from 2.8 to 21.7 pg/mL. A more recent study by Sauerwein et al. (2000) measured fluctuations in plasma E2 in Simmental breeding bulls with an average age of 8.4 years with and without recombinant bovine somatotropin (rbST) treatment. Untreated controls (injected with vehicle only) and treated bulls exhibited fluctuations in E2 concentrations over the 25 week study. Concentrations of E2 in untreated bulls ranged from approximately 5 pg/mL to approximately 23 pg/mL, based on Figure 2 in this paper, with a mean pre-injection concentration of 12.0 ± 1.5 pg/mL. No papers were identified which discussed E2 concentration in young, growing bulls; however, given its possible role in normal development, increased levels of E2 in growing bulls of high genetic merit may be expected to have slightly elevated levels. Based on these ranges and those established by the reference laboratory, E2 concentrations of 24 pg/mL are not a concern, and should be considered within normal fluctuations for bulls.

Analysis of the hematological parameters for all of the clones and comparators (Chart E311) showed no remarkable findings. Sixteen of the 17 analytes measured for clones were within the range of the comparators, or had only one difference. No problems were identified with red blood cell measurements (e.g., anemia or polycythemia (increase in the total mass of red blood cells in the body)), or white blood cell problems (e.g. leukocytosis (increase in WBC count) or leukopenia (low WBC count)) were seen. Two animals (Clones #99 and #108) presented with a MCV below that of the comparators, but given the lack of corresponding evidence of anemia in these animals, these values have no clinical significance. Clone #108 was culled due to dwarfism.

(j) Animal Analysis

Unless specifically mentioned, no differences were observed between an individual clone's clinical chemistry or hematology values and the range presented by the comparators.

Clone #103 (Red Angus, 6.5 month old heifer) exhibited elevated potassium (K+), asparagine transferase (AST), and creatine kinase (CK) levels relative to the comparators. None of the values in the hemogram exceed the range defined by the comparators. A physical exam conducted on the same day as the blood draws showed no abnormalities. The sample drawn from this calf had the highest hemolysis index (353) of all of the samples, indicating poor sample handling. Potassium is an electrolyte found mostly within the cell, and its elevation could be caused by sample hemolysis. Asparagine transferase (AST) is an enzyme normally found in liver or muscle tissue that can be released when liver or muscle is damaged. Creatine kinase is a muscle specific enzyme that is released when there is muscle damage. Although these values are elevated, they are low enough to imply only minor muscle damage, similar to that observed when an animal is recumbent for an extended period of time, struggling in a squeeze chute, or may be artifactual due to blood sample handling leading to hemolysis. Given that this animal exhibited no clinical abnormalities, even if these analyte levels are not due to artifact or poor sample handling, these changes would likely have no clinical significance.

Clone #108 was an Angus heifer that is characterized as a dwarf with frequent bloat. She was reported to have been severely deformed with abnormal gastro-intestinal (GI) motility. Interestingly, clinical chemistry results show only a decrease in TIBC compared to the comparator group. The hemogram indicated high RBC and slightly lower MCV relative to comparators. The hematocrit was within the range of the comparators. Despite the animal's obvious physical abnormalities, laboratory values were not significantly out of range.

(k) Conclusions for 6-18 Month Group: Animal Safety

Clones in this age group exhibited no differences from comparators with respect to their overall health, and were indistinguishable from the comparator group on the basis of clinical and laboratory tests. No residual health problems were noted in this group of animals, with the exception of the dwarf calf that was identified visually and culled. Based on these observations, there are no apparent health risks in this age group of animals.

(l) Conclusions for 6-18 Month Group: Food Safety

No material differences were observed between the six to eighteen month old clone cohort and their approximately age-matched, similarly reared comparators. In fact, the clones are indistinguishable from the comparators, and thus would be unlikely to pose any risk for consumption as human food or in animal feed above conventional animals now in the food supply.

5. Charts and Tables

The following Charts summarize the results of laboratory data derived from the Cyagra clones and corresponding non-clone comparators. In Charts 100, 102, 110, 112, 200, 202, 212, 300, 302, 310, and 312, the clones' laboratory data were compared to the Cornell veterinary laboratory data and to the approximately age- and breed- matched comparators' data. A laboratory value from a clone that exceeded the range of the Cornell Reference Range or comparators was initially flagged regardless of how much it was out of range. After all of the comparisons had been made (i.e., clones to comparators, clones to reference range, and comparators to reference range), it became apparent that the most appropriate comparison for clinical relevance was clones to comparators. We then determined the clinical relevance of the out of range values. Clinical relevance was defined as laboratory value observed in the clones that was more than 10 percent out of the comparator range, or, one that based on veterinary clinical judgment, was likely to cause concern. The published literature was consulted for non-standard clinical chemistry endpoints such as IGF-I and estrogen. Laboratory values determined to be not clinically relevant are represented in the Charts as gray boxes. Clinical relevance is presented in charts 101, 111, 201, 211, 301, and 311.

Footnotes

1Cyagra Inc. is a privately held biotechnology company commercializing SCNT technology for the agricultural sector.

2A hemogram is a panel of measurements characterizing the nature of the circulating blood in an animal or human.

3Cryptorchidism is undesirable because of its heritability, its adverse effect on fertility, and potential for the development of testicular cancer in animals living long enough to allow neoplasia to develop. From a veterinary standpoint, however, testicular neoplasia is more of an issue with companion animals, as they are generally longer-lived than farm animals.

4It should be noted that because the reference range represents only 95 percent of the animals used in its derivation, even comparison of the animals used for the derivation will not fit exactly within the distribution. Thus, if the reference range were expanded to include those values outside the 95 percent distribution, it is likely that the clone and comparator populations would show a higher degree of "fit" than is observed in this analysis.

5Rotavirus is a common enteric pathogen in cattle to which many calves are exposed; some succumb. There is a commercial vaccine for the dam to increase immunity to rotavirus in the colostrum to passively immunize calves in order to protect them.

6Metabolizable energy is an estimate of the number of calories absorbed by the animal after digestion.

The Cyagra Dataset (pdf)
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