On May 21, 2003, members of the CVM Animal Biotechnology Working Group Animal Clone Subcommittee held a teleconference with members of three companies engaged in animal cloning to discuss their veterinary care protocols for pregnant surrogates and perinatal animal clones. The purpose of this call was to obtain information on risks that they had identified in the course of their cloning practices that might not be presented in the publicly available literature so that CVM can ensure that the Center has been comprehensive in its risk assessments of food and animal safety. Unless otherwise noted, the species discussed was bovine. The following are minutes of the conference in which the names of individuals and their firms have been redacted.

Firm 1 began the discussion by outlining their prenatal care regimens. They generally start monitoring surrogate females 30 days after embryo transfer by ultrasound, initially to confirm pregnancy, and to count fetuses, because two embryos are usually implanted into each female. Monitoring is then conducted every 30 days. Most pregnancies spontaneously abort before 90 days gestation. Very few individuals carry pregnancy as far as 150 days. Typically, there are no adverse health indications in recipients prior to 150 days of gestation. After 150 days, animals are monitored daily and more closely. Herdsmen are trained to watch animals for abdominal size, an indicator of developing hydrops, and veterinary exams are more frequent. Palpation can reveal if there is excess fluid in the uterus. The firm noted that they have found that hydrops is not an all or nothing condition. Some pregnancies have a small to moderate amount of excess fluid, but this does not necessarily place the dam or fetus in danger. Many pregnancies go to term, and there may be more fluid than expected at delivery, but the calf and cow are fine. It is a matter of degree, and a judgment call for the veterinarian. If hydrops becomes severe, the pregnancy is terminated with prostaglandin and dexamethasone injection at the discretion of a veterinarian. Since they started monitoring for hydrops, there have been no death losses among the surrogates. If a surrogate carries a pregnancy beyond 150 days, she is not used as a recipient again.

Firm 2 indicated that their findings are similar to Firm 1. Hydrops is a matter of degree, and their veterinarian refers to it as “pseudo-hydrops” when there is only a small to moderate amount of excess fluid. The only identified issue for surrogates is hydrops. Most of their calves are delivered by planned Caeserian-section, so dystocia is not an issue. However, Caesarian delivery poses its own set of risks.

Firm 1 added that they have observed a higher death loss during C-section when there is excess fluid, but this has occurred in very few animals. In response to a question from CVM inquiring about relative frequencies of adverse outcomes in IVF and SCNT, Firm 1 stated that they have produced 500 – 600 IVF calves and calves from in vitro cultured embryos. Many of the same outcomes such as LOS, are observed in clone pregnancies as in IVF pregnancies, but the frequency is lower now than in the past. They have observed some hydrops in IVF pregnancies, changes in placentation, and umbilicus. There is a higher incidence of these changes in SCNT compared to IVF. The firm stated that these problems with IVF have decreased in incidence with the use of improved culture conditions.

Firm 2 indicated that for IVF, calving generally occurs at the customer’s farm, and that they haven’t had any reports of dystocia. Because they use heifers as recipients, and the high values of the offspring, their vets prefer to do C-section on the recipients. They have allowed some to go through natural birth, and parturitions were normal.

CVM asked whether any blood variables have been identified that might be predictive of hydrops or LOS. Firm 2 replied that no recipient blood variables have been correlated with pregnancy outcome. Currently there is no reliable way to predict LOS prenatally.

In response to a CVM question of how LOS is diagnosed, Firm 1 replied that they rely on the experience of a veterinarian, who can “feel” if a fetus has LOS. They have tried to score mammary development and rectal palpation of the uterus to determine the amount of fluid present. Other methods are not used as umbilical vessels are not enlarged. Generally, if the recipient is going through normal late pregnancy changes, the calf will likely be normal. They feel that it is a subjective judgment based on clinical impression. Firm 2 added that their veterinarian is using mammary development and palpation, but does not record the findings.

In answer to CVM’s question as to whether there were differences between IVF vs. SCNT-derived animals, Firm 1 answered that the potential problems were similar, but there are less LOS in calves derived from IVF. Firm 2 added that they were not concerned about LOS and even deliver some calves naturally.

In response to question about therapeutic interventions and equipment available at calving, Firm 2 replied that oxygen and an IV drip are available, and blood glucose and blood gas-pO2 are monitored. Firm 2 stated that they rely less on pO2 now than they used to, as they are now getting enough normal calves that pO2 is not routinely measured. If the placenta and other factors are normal, pO2 is not monitored at all.

Firm 2 stated that it is likely that they over-react and are overly protective of these calves because they are so valuable. There was a general consensus among all of the producers regarding the very high degree of observation and care that is given these animals, and whether such concern was necessary.

Firm 3 agreed that their observations have been similar. All their calves are taken by C-section. There tends to be a focus on detecting the potential negative outcome—but a low percentage of recipients are affected with hydrops. They generally don’t see any problems until about day 230 – 240 of gestation. Then they see a good percentage of the recipients become ketotic. These animals are treated them with propylene glycol, dextrose drip if necessary, or, if it is severe ketosis, by transfaunation (transferring rumen material from a healthy cow). They stated that there is a correlation between severe ketosis of the dam and calf thriftiness—the calf requires more care and may spend more time in the hospital. On average, SCNT calves spend two to two and half weeks under close observation. They also have found no correlation between blood variables and outcome. Outcomes seem to be cell line dependent. They tend to see the same problems within a cell line used as a donor source.

Firm 2, in response to a question as to whether any of their female clones had calved replied that have one female that has calved and is currently lactating. Firm 1 has several, some are in their second lactation, but almost all of them are transgenic.

In response to a question about umbilical abnormalities in clone calves, Firm 3 said that more than 50% of calves have an enlarged umbilicus requiring surgery. The cord doesn’t dry up and drop-off/heal-over. Umbilical extirpation reduces risk of infection. This does not appear to be related to cell line. Firm 2 said that they see some of the same problems, and routinely remove the umbilicus to decrease the risk of infection.

In response to question about how embryos are transferred to recipients, Firm 3 stated that they use non-surgical transfer in cattle, but surgical transfer in swine. Generally, 50 – 70 embryos are transferred into each gilt. In response to question whether there were any similarities in complications between cattle and swine, Firm 3 answered that there are no similarities between swine and cattle. One-cell NT embryos are transferred into swine, and those embryos spend very little time in culture. Pregnant gilts are monitored weekly to look for normal fetal development up through day 35, and normal bone structure up through day 40 – 45. They have observed agalactia in sows, as well as some placentation issues, but no hydrops or LOS.

Firm 2 stated that another firm involved in cloning has also stated that they see no LOS or umbilical problems in pigs, but that recipient gilts have slightly longer pregnancies (by a few days). That firm gets 5 – 6 pigs per litter. Recipient dams farrow normally, and labor initiates normally.

In response to question on frequency of hydrops, Firm 1 answered that their vets have only terminated one or two pregnancies per year for hydrops, out of 250 – 300 clone-bearing pregnancies.

The teleconference concluded with CVM updating the animal clone producers on the status of the food consumption and animal risk assessment, and extending the sincere thanks of the Center for the gracious and extensive cooperation and help that has been offered by the producers.