# Presentation: 254915

## Animal Safety

- Amey L. Adams, PhD
- Center for Veterinary Medicine
- Food and Drug Administration
- November 4, 2003

## Context for Consideration of Animal Health Risks

- Do the risks experienced by animals involved in the cloning process differ qualitatively from those experienced by animals undergoing other assisted reproductive technologies?

**Notes:**
Evidence indicates that cloning increases the risks experienced by the animals involved compared to natural mating, namely to the clone animals themselves and the female animals used as surrogates.  But are these risks different from or more frequent than those experienced by animals involved in other assisted reproductive technologies such as artificial insemination or in-vitro fertilization?

## Critical Biological Systems Approach: Animal Safety

**Perinatal**
**Development **
**and Function**

**Reproductive Development**
**And Function**

**Post-Pubertal**
**Maturation**

**Cell Fusion Through **
**Fetal Development**

**Progeny**** **

- Development of hydrops, normal expulsion of non-viable conceptus, return to cycling, reproductive tract exams

- Birth weight, evidence of hydrops or dystocia, dam’s readiness for delivery, onset of lactation and mothering behavior, newborn organ function and IgG levels

- Onset of puberty, estrous cycling and behavior, breeding soundness, sperm motility and morphology, reproductive tract exams, hormone profiles, breeding success, delivery of healthy offspring

- Growth to maturity, gait, activity level, blood clinical chemistry, heart and lung function

- Growth and development, feed intake, activity level, blood clinical chemistry, heart and lung function, disease incidence

**Surrogate Dam and Fetus**

**Surrogate Dams and Newborn Clones**

**Breeding Clones**

**Juvenile Clones**

**Adult Clones**

**Juvenile**
**Development **
**and Function**

**Notes:**
The approach we used was similar to the Critical Systems analysis used for the food safety evaluation, but with different endpoints in mind.  Node 1: we primarily focused on mid- to late gestation, as this appeared to be the period of greatest risk to the dam, and set the stage for the neonatal period for the clone animal.  The data we looked for included such late gestational complications as hydrops, dystocia, and whether there were any reports of surrogates retaining non-viable fetus or fetal membranes following a failed pregnancy.  Node 2: we searched available references for data related to Large Offspring Syndrome and associated complications, as well as indications of the health of the dam.  Nodes 3/4: during this developmental node, we searched for data that related to the health and development of the young clone, both pre- and post-weaning.  Nodes 5&6 are simultaneous observations of the clones’ continued viability and development through puberty and into adulthood.  Data for these nodes are extremely sparse.  As with progeny, some data has been extrapolated from mice to try to answer whether or not there are health risks for older clones and their offspring.

## Overview

- Surrogate Dams of Clones
- Neonatal Clones
- Juvenile to Prepuberty Clones
- Puberty through Reproductive Maturity
- Maturity and Aging
- Progeny of Clones

**Notes:**
So, in short, these are the lifestages we considered as we analyzed the data...

## Surrogate Dams

## Cattle and sheep:

- Studies indicate increased risk of mid- to late- gestational complications
- Hydrops (edema of fetus or fetal membranes)
- Dystocia (difficulty giving birth)
- Other complications

**Notes:**
As we sorted through the literature, it became apparent that different outcomes were experienced by different species.  Cattle and sheep surrogates experienced a number of mid- to late gestational complications, some mild, some severe enough to result in death or permanent injury to the animal. These complications have also been reported in surrogate cows and ewes carrying IVF (test-tube) pregnancies.

## Hydrops

- A collective term referring to excessive build-up of fluids in fetus or fetal membranes
- May be detected as early as the sixth month of pregnancy in cattle (clone producers)

**Notes:**
Hydrops occurs rarely in natural and AI pregnancies at a rate of ~1/7500 pregnancies in cattle.  One early report of a commercial enterprise estimated the incidence of hydrops in cattle carrying IVF pregnancies was 1/200 pregnancies, or ~0.05%.

Because the condition is so rare in the general cattle population, we realized that very few veterinarians would have experience dealing with a hydrops pregnancy.  For this reason, we decided to contact some animal cloning companies to learn more about the pathology of this condition from their veterinary staff.

## Hydrops (continued)

- Degree varies from mild to severe (clone producers)
- Severe hydrops - death of the dam and/or fetus (Heyman et al. 2002)
- Incidence highly variable among laboratories
- Data indicate risk higher in clone pregnancies vs. IVF (Pace et al. 2002 (some transgenic; 30/178), clone producers (~1/300))

**Notes:**
Those we spoke with indicated that hydrops did occur at a higher rate in clone-bearing pregnancies compared to IVF, but the condition did not always progress to a point where the cow or the developing fetus were at risk.  However, the literature indicates that if the condition does progress and the pregnancy is not terminated, it will most likely result in the death of the fetus and possibly also the dam.

Review of the literature and discussion with the companies revealed that incidence of hydrops was highly variable among laboratories.  The Pace et al. study indicated that in cows carrying clone pregnancies (some of them transgenic) beyond 60 days, the incidence of hydrops was ~17%.  By comparison, one of the clone producers indicated that incidence of hydrops at their lab was ~1 in 200 or 300 clone-bearing pregnancies.

## Dystocia

- Causes:
- fetal oversize compared to pelvic opening
- malpresentation of the fetus
- multiple fetuses presenting simultaneously
- Also in conventionally bred animals bearing single, large fetus (Lucy 2001, Dwyer 2003)

**Notes:**
Difficult labor is a problem experienced by all reproducing mammals.  It is most commonly caused by a large fetus trying to pass through the dam’s pelvic opening, but other complications can arise to cause dystocia.  In conventionally bred dairy and beef cattle, the overall rate of dystocia is between 4 and 6%, and tends to be higher in heifers.  The rate is higher in ewes, ranging from 10 to 30%, and varies among breeds.

## Dystocia in Clone Pregnancies

- Often result from fetal oversize (i.e. Large Offspring Syndrome)
- May cause damage to the reproductive tract and musculo-skeletal system
- Incidence varies with LOS, management decisions (clone producers)

**Notes:**
In clone-bearing pregnancies, dystocia is most often associated with LOS.  Dystocia may result in retained placenta, uterine infections, permanent damage to the reproductive tract, and musculo-skeletal damage.  Such complications could compromise future reproductive capability and result in culling of the dam due to reproductive failure or other injuries.  The incidence of dystocia varies with incidence of LOS, a topic we will discuss in greater detail in relation to the neonate.

## Less Frequent Complications

- Also observed in cattle and sheep surrogate dams (Hammer et al. 2001, Wells et al. 1998 & 1999, Ptak et al. 2002):
- Poor or absent mammary development
- Absent or atypical signs of labor (uterine inertia)
- Agalactia (failure to lactate)
- Impaired maternal behavior
- Frequency of these anomalies were not reported

**Notes:**
Other less frequent complications have been observed.  These do not necessarily indicate a risk to the dam, but could increase the risk to the neonatal clone.  For example, failure to lactate could have implications for transfer of immunity through colostrum, and neonatal nutrition.  Also, it is important for the dam to interact appropriately with the neonate immediately after birth in order to stimulate it to breathe, stand and nurse.

## Goats and Swine

- No reported complications in surrogate goats
- No reports of hydrops or LOS in either species
- Some reports indicate lack of mammary development, agalactia, uterine inertia in swine (Carter et al. 2002 (transgenic), clone producers) (Frequency not reported)

**Notes:**
Reports indicate fewer complications for surrogate dams of the other two livestock species which have been cloned.  In summary, there is no indication of increased risk of morbidity or mortality for the surrogate dams of these species.

## Neonatal Clones

## Cattle and Sheep

- Large Offspring Syndrome (LOS)
- Reported in IVF, BNT, SCNT-derived cattle and sheep (Kruip and de Daas 1997, Knight et al. 2001, Chavatte-Palmer et al 2002, Ptak et al. 2002):
- Fetus or newborn >20% above average weight
- Respiratory complications
- Internal organ defects (heart and kidney)
- Musculo-skeletal defects
- Poor or absent suckle reflex

**Notes:**
Large Offspring Syndrome was first noted in calves and lambs derived by IVF, but has since also been noted in both embryonic and somatic cell clones.  LOS is named for its most obvious sign: an oversized fetus or newborn.  However, a number of other co-morbidities have been noted in association with fetal oversize.  The most common of these is respiratory distress due to a variety of causes, including failure of the lungs to inflate, pulmonary hypertension, inhalation of amniotic fluid and subsequent pneumonia.  There may also be musculo-skeletal or internal organ defects, notably defects of the chambers or valves of the heart, immature or cystic kidneys, tendon contracture, and joint or skull malformations.  Calves and lambs with LOS also have been noted to be slow to stand and have poor suckle reflex.

## LOS (continued)

- Increases risk of dystocia
- Dystocia - stress neonatal calf or lamb, increase risk of mortality and morbidity (Moore et al. 2002, Kato et al. 1998):
- Premature separation of placenta
- Inhalation of amniotic fluid - respiratory complications

**Notes:**
It can be difficult to distinguish complications of LOS which are directly related to embryo manipulation, and those which are indirectly related due to crowding in the uterus and fetal distress during dystocia.  LOS increases the risk of dystocia because the fetus is often too large to pass through the pelvic opening without assistance.  During dystocia, the placenta may separate prematurely, depriving the young animal of oxygen and possibly forcing it to try to breathe prematurely.  Inhalation of fluid predisposes the calf to respiratory illness such as pneumonia.

## LOS continued

- Some complications reversible, depending on severity
- Incidence variable among labs for SCNT (8 to 50%) (Miyashita et al. 2002, Kato et al. 2000)
- LOS in IVF calves 7 to 31% (Hasler et al. 1995, Kruip and den Daas 1997)

**Notes:**
Evidence indicates that many calves and lambs survive despite LOS.  Complications such as flexor tendon contracture and some respiratory problems are amenable to therapy, and suckle reflex may establish on its own.

Like hydrops, incidence of LOS is highly variable among labs, although the incidence of LOS is higher than the incidence of hydrops.  Numerically, the range reported in the literature is similar, though higher in clone animals than animals derived by in-vitro fertilization.

## Neonatal Survival

- Critical survival period first 48 hours (Cyagra)
- Neonatal death rate ~ 20% in clone cattle (Pace et al. 2002 (some transgenic), Cyagra)
- Death rate in early IVF cattle studies 14 to 16% (Hasler et al. 1995, Kruip and den Daas 1997)

**Notes:**
According to the data provided by Cyagra, and conversations with this firm, the most critical period is during the first 48 hours after birth.  The majority of animals (as much as 89%) that survive this period appear to develop normally.

Early neonatal death rate is numerically higher among clone calves compared to early reports of IVF calves.  There is no way to be certain if this difference is statistically significant, but smaller studies which have compared IVF and SCNT under the same conditions indicate that neonatal morbidity and mortality is higher among clones compared to IVF.

## Neonatal Goats and Swine

- Two reports of neonatal mortality in goat clones (Keefer et al. 2001 (1/6), Keefer et al. 2002 (2/9))
- Few reports of complications in neonatal swine clones
- Low birth-weight (Polejaeva et al. 2000, Walker et al. 2002) (actual frequency not reported)
- One report of physical deformity (anal atresia) in non-transgenic clone pig (1/28) (Walker et al. 2002)

**Notes:**
There is very little quantitative data on neonatal clones of swine and goats.  In general, it appears clones of these species experience few complications or health problems, and survival rates are generally high.  The Keefer goat studies indicated that two goat kids died during delivery (both of these were twins to surviving clones) and one died of a respiratory infection.  Two studies have reported low-birth weights in neonatal clone swine, but both reports indicated pigs were otherwise healthy.  One study reported a piglet born with anal atresia (absence of an anal opening).  All of these problems have been noted to occur in conventionally bred goats and swine.

## Juvenile to Prepuberty

## Juvenile Clones

- Most reports indicate normal growth and development following the neonatal period
- Behavioral studies in cattle and swine note no abnormalities (Savage et al. 2003, Archer et al. 2003)
- Results of blood tests indicate cattle and swine in this age group are mostly within the range of conventionally bred animals of the same age (Cyagra, Archer et al. 2003; Chavatte-Palmer et al. 2002, Kato et al. 2000)

**Notes:**
Available data indicate that, for the most part, animals that survive the critical neonatal period grow and develop normally.  Blood tests and behavioral studies reveal no subtle abnormalities.

## Juvenile Cattle

- A few individual animals reported to have health problems which may stem from LOS (Cyagra, n=134)
- Non-resolving musculo-skeletal (2)
- Failure to thrive (1)
- Umbilical surgeries (29)
- GI tract problems (2)
- Heart abnormalities (1)
- Some of these animals had more than one anomaly

**Notes:**
A few calves, however, continued to demonstrate abnormalities which, in most cases, appear to be the result of neonatal complications which did not resolve.  The Cyagra report indicated one calf with flexor tendon contracture that did not respond to therapy, one calf with bone deformities, one calf was diagnosed with “failure to thrive”, one calf with recurring bloat and one with poor GI tract motility, and one calf with a heart murmur...The most commonly reported problem in this group of calves was enlarged umbilicus, sometimes with patent blood vessels.  Twenty-nine calves received umbilical surgery.

We could not identify reports on sheep in this age group.

## Juvenile Goats and Swine

- One report of respiratory infections in growing goat clones (2/6) (Keefer et al. 2001)
- One report of hyperkeratosis in a swine clone (1/9) (Archer et al. 2003)

**Notes:**
In goats and swine, a few incidents of health problems.  May be related to management. Respiratory infections are a common problem in conventionally bred goats.  Hyperkeratosis (or parakeratosis) is rare, but is generally related to nutrition.

## Other Complications

- Cryptorchidism (3/134) (Cyagra) (all from one cell line)
- Dwarfism (1/134) (Cyagra)
- Hyperkeratosis (Archer et al. 2003) (1/9)
- Cryptorichidism and dwarfism may be related to genetics of nuclear donor
- Hyperkeratosis (dermatitis vegetans) rarely a recessive gene

**Notes:**
Other complications may be related to genetics of the nuclear donor.  Cryptorchidism and dwarfism occur in conventionally bred cattle, and appear to be recessive traits.  I mention hyperkeratosis here again because, although it is usually related to dietary problems, it is on rare occasions associated with a recessive gene.

## Puberty through Reproductive Maturity

## Pubertal Cattle

- No health problems noted; blood chemistries normal (Cyagra)
- Most reports indicate heifers reach puberty, conceive and deliver healthy calves (Lanza et al. 2001 (transgenic), Pace et al. 2002 (some transgenic), Enright et al. 2002)
- One report of clone heifers achieving puberty slightly later than controls, but within the normal range for their breed (actual frequency not reported) (Enright et al. 2002)
- No detailed information on bulls (Forsburg et al. 2002, Kato et al. 2000, Kubota et al. 2000)

**Notes:**
The reproductive system is the most sensitive to derangement, and thus is an important indicator of possible underlying health problems.  The few studies that have followed cattle through reproductive maturity indicate that female cattle clones are able to reproduce normally.

Only one report, based on four heifers, was sufficiently detailed, and indicated that these animals achieved puberty at a later age and heavier weight than their counterparts, but within the normal historical range for their breed.  Only one of these heifers failed to conceive to AI.

No details are available on sexual development of bull calves.  Forsburg et al. reported that Gene has produced semen (???)

## Other Species

- One report each on fertility of ram and buck clones, indicating normal age at puberty, fertility, and sperm quality (Wells et al. 1998, Gauthier et al. 2001)
- One report of goat doe clones conceiving and delivering normal offspring (Reggio et al. 2001)
- No reports on reproduction in swine clones

**Notes:**
Data on reproduction in clones is even more sparse in other species.  No abnormalities have been reported.

## Maturity and Aging

- Insufficient time to evaluate aging in farm animal species
- Aging study in mice observed shorter lifespans, increased health problems (Ogonuki et al. 2002)
- Studies of telomeres conflicting; not good predictor of lifespan (Betts et al. 2001, Lanza et al. 2000, Tian et al. 2000, Miyashita et al. 2002, Kubota et al. 2000)

**Notes:**
Not enough time has elapsed to evaluate the health of clones as they mature and age.  

The only information available on ageing in clones comes from one study of mice, which indicated that clones experienced shorter lifespans and serious health problems as they aged. 

Some scientists have suggested that telomere length might be used as an indicator of ageing.  However, studies of telomere length in clone livestock have yielded conflicting results.  It was reported that Dolly’s telomeres were the same length as her six-year old nuclear donor.  Other reports, however, have indicated that telomeres in cattle clones were the same length or longer as their age-matched comparators.

## Offspring of Clones

- Several cursory reports state offspring are “normal and healthy”
- No details available in farm animal species
- Reports from mice indicate offspring do not inherit clone-associated abnormalities of parents (Shimazowa et al. 2002, Tamashiro et al. 2002)

**Notes:**
There have been no detailed studies of progeny of livestock clones.  Studies of animal clones report only that their offspring are normal and healthy.  Information from mouse studies indicate that progeny of clone mice do not inherit the abnormalities exhibited by their parents.

Shimazowa et al. bred abnormal clone mice (oversized at birth, born with eyes open and abnormally large placentas) and obtained normal size litters of apparently normal offspring (normal size, born with closed eyes and normal placentas).

Tamashiro et al. reported that clone mice developed obesity by 8 weeks of age.  However, their offspring were of normal weight compared to age-matched control mice of the same breeding background.

## Conclusions

## Cattle and Sheep:

- Critical period: late gestation - first post-natal days (surrogate dams and clones)
- Risk of perinatal morbidity and mortality higher in clone pregnancies compared to other ARTs
- Most clones that survive perinatal period appear healthy and similar to conventional counterparts
- No abnormalities reported in offspring

**Notes:**
The majority of the available evidence indicates that clone cattle and their surrogate dams are at increased risk of morbidity and mortality compared to other forms of ART.  Although research in sheep is limited, the results are consistent with those for cattle, indicating that this species is also at elevated risk.  For this reason, these two species have been grouped together in the consideration of relative risk.

Late gestation through early post natal is the period of greatest risk for these animals.  Incidence of morbidity and mortality during this period is higher than IVF, even during the early development of that technology.

Following the perinatal period, clones generally grow and develop normally.  The few reports of morbidity and mortality in cattle clones during juvenile development appear to be related to problems experienced in the neonatal period which failed to resolve.

There are no details available on offspring of clones, although they are reported to be healthy and normal.

## Swine

- A few reports of complications in surrogate sows
- One report of deformity in a clone pig
- Anal atresia also noted in conventionally bred swine
- No reports on reproductive maturity of clones or health of offspring

**Notes:**
There have been some reports of agalactia and uterine inertia in surrogate sows.  These complications are not necessarily a risk to the dam, but may represent a risk to the piglets if labor does not proceed normally and the sow does not lactate.

While there have been some reports of deformities and health problems in transgenic clone swine, only one report was found of a deformity in non-transgenic clone swine.  Anal atresia is known to occur in conventionally bred pigs.

## Goats

- No complications noted for surrogate does carrying goat clones
- Very few health problems noted in goat clones
- Two deaths during labor, three deaths due to respiratory infection
- Appear to mature normally and produce healthy offspring

**Notes:**
There have been no reports of pregnancy complications for surrogate goats.  In general, pregnancies which last beyond the first 40 days progress normally.  Given the small numbers of animals studied in this species, it cannot be determined whether young goat clones are at greater risk of illness or death than non-clones. However, there have been no reports of hydrops or LOS in clone goats, in contrast to sheep and cattle.

## Overall Conclusions

- No qualitative differences from other ARTs or natural breeding
- Frequency of anomalies increased even relative to IVF
- Adverse outcomes observed more frequently in cattle and sheep than swine and goats

**Notes:**
The complications observed in clone-bearing pregnancies have been noted in other forms of ART as well, albeit at a lower frequency compared to cloning.  The weight of the evidence indicates that cattle and sheep clones and their surrogates are at greatest risk of morbidity and mortality compared to other species which have been cloned.  In comparison, very few reports of serious illness or death exist for goats and swine.

## VMAC Question

- Question 1:  Based on what we have presented, has the risk assessment adequately identified the hazards and characterized the risks relating to animal health?