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Safety & Health

CVM Memorandum II - ViaGen Industry Meetings

April 25, 2005

ViaGen industry meeting

From CVM: From ViaGen:

Amey Adams Irina Polejaeva Cecilia Aquila Shawn Walker Mary Bartholomew Rial Christensen Siobhan DeLancey Eric Dubbin Kevin Greenlees Chris Horstkamp John Matheson Julia Oriani Larisa Rudenko Tim Schell

Larisa Rudenko called the meeting to order and outlined that the goal of the meeting was to further understand

  • The overall study design;
  • The nature of the data that Viagen has shared with CVM; and
  • What information is still required to be able to interpret Viagen’s data

Irina Polejaeva gave a brief overview on cloning in general and ViaGen’s use of cloning in particular. In response to a question, she stated that ViaGen is currently using surgical implantation of clone embryos into surrogate mothers, but that they hope to refine the technology to achieve the same results with non-surgical implantation.

Dr. Polejaeva also noted that ViaGen now owns a group of sheep clones purchased from PPL that are now older than Dolly was when she was euthanized. It is ViaGen’s goal to publish on the status of these clones when they reach ten years of age. Some of the sheep are also transgenic.

Dr. Rudenko noted that these kinds of data would be very useful in CVM’s risk assessment and asked that if possible, ViaGen supply any available information on the dates of birth of these animals and a brief health update on the sheep.

In explaining the swine cloning project, Dr. Polejaeva stated that ViaGen has been more successful in producing litters of piglets when embryos are cultured for five days and then 20-25 blastocysts implanted. This usually results in a litter size of approximately 8 piglets. By contrast, if embryos are transferred immediately, generally only 20 percent will still be viable at the blastocyst stage. Dr. Polejaeva stated that the optimal number of blastocysts to be implanted for large litters is unknown, but she theorized that implanting a high number of blastocysts may actually limit successful implantation and gestation. The animals produced in Athens, GA, and used in this study were the products of embryos that were cultured for one day prior to implantation.

With the five-day culture protocol, ViaGen has been as successful in producing piglets with cloning as it has with IVF. Dr. Polejaeva also noted that surrogate pigs do not need to be of the same breed as the donor.

Dr. Polejaeva and Dr. Walker then answered questions regarding Experiments 1 & 2, also known as Food Safety Studies 1 & 2.

Experiment 1: Boar Clones and Comparators

Experiment 1 consists of five boar clones and fifteen comparators. Clones 18, 19, and 20 are from the same cell line, while clone 22 is from a different cell line. Clone 24 is a Duroc, while all the others animals in the study are from Hampshire lines.

All clones were delivered by Caesarian section at the conclusion of a conventional gestation period (105 days). However, Dr. Walker noted that swine clones appear to need increased time in the womb, and that the piglets delivered appeared to be the developmental equivalent of neonates that were 14 days premature. ViaGen noted that the gestation of clones tends to be slightly longer than that of conventional animals by a few days. They also noted that it is difficult to induce labor of a litter of swine clones on the predicted day of parturition, although they can be induced two or three days after the predicted parturition date. The piglets in Experiment 1 had low birth weights, and were subsequently colostrum-deprived and bottle fed. They were initially kept in a pathogen-free environment but were moved to a piggery and were castrated at about three to four weeks of age. By comparison, the barrow comparators were delivered vaginally, were allowed to nurse colostrum, and were kept with the gilts. They were castrated at three to fours days of age.

Clones and comparators were fed the same diets post-weaning.

In comparing the data, Dr. Polejaeva pointed out that some of the comparators that died were not included in the data collection. Conversely, some of the barrow clones were kept alive beyond a point when they would have been commercially viable, simply because they were clones. In other words, some of the barrow clones received medical care that they would not have received in commercial settings, and were sustained because they were clones. In response to a question, Dr. Polejaeva indicated that there was no genetic relationship between these barrows and the cloned sires used to produce offspring in Experiment 2. Experiment 1 is primarily for carcass evaulation. This experiment will not provide valid comparisons between clones and non-clones for health observations and growth comparisons because of the differences in farrowing (vaginal v. C section), husbandry (colostrum deprivation in clones, late castration, SPF differences), and gestational age (clones delivered prematurely).

Experiment 2: Progeny of Cloned Boars and Comparators

The boar clones were raised, and their semen collected and chilled, at Prolinia in Athens, GA. Comparator boars were kept and collected elsewhere. Collection procedures were the same for all boars and utilized the same semen extenders and shippers.

Three of the boar clones (002, 003, and 005) were copies of H498, a five-year-old boar also used to produce progeny for the study. These boar clones were first used for breeding at approximately seven to eight months.

Another boar clone (007) was derived from a second boar (5001); boar 5001 also produced two sons (18128 and 25515). Boar 5001 was not available for breeding at the time of the study and so his sons were used as sires in experiment 2. The sons of 5001 were approximately ten months old at the time of breeding.

Gilts used in the study were from the Clay Center Research Facility and were from six or seven different sire lines. They were selected on the basis of six different qualities based on sire genetics. All gilts were inseminated and farrowed at Clay Center. AI was performed at 12 and 24 hours past the detection of the onset of estrus.

No incidence of Large Offspring Syndrome (LOS) was noted in any of the clones, comparators or progeny. There was some incidence of “runt” piglets, but no more than would be expected of a conventional litter.

Although the boar clones in Experiment 1 were delivered via Caesarian section, the progeny of both clones and comparators were born conventionally: that is, the gilts were allowed to initiate labor, delivery, and lactation. It was noted that the gestation of clones tends to be slightly longer than that of conventional animals by a few days. It was also noted that it is difficult to prematurely induce the labor of a litter of cloned piglets.

Progeny of clones and comparators both initiated respiration on their own without intervention, although handlers were advised to intervene in the parturition of any litter if more than five minutes passed between presentation of piglets, whether or not the gilt appeared to be in distress. A visual exam of placentae revealed no abnormalities in either clones or comparators.
Update: Dr Jodi Sterle is the contact for the cloned pig placenta and she is willing to discuss her observations. Her number is 979-845-7616.

It is unknown how the progeny of clones and comparators were sorted in pens following weaning. Dr. Polejaeva indicated that she would contact the staff at Clay Center to obtain this information.
Update: According to Ted Atkins at the Clay center, the piglets were kept separate based on litters through the nursery but were then mixed during growing and finishing.

In her presentation, Dr. Polejaeva listed the parameters that this study would be able to assess, namely:

  • Production characteristics of clones v. non-clones 4
  • Reproduction parameters including semen quality, farrowing rate, and litter size
  • Offspring parameters for 625 animals including birth weight, growth rate, animal health, blood chemistry, carcass data, and meat composition

Clarifications in Data Collection

There were some questions about the abbreviations used in the data. Dr. Walker said that he would forward a list of abbreviations and definitions to us.

Update: This document is now on the O drive at O:\Individl\ONADE\DeLancey\ViaGen\Abbreviations.doc.

OVERLAY= Piglet crushed by dam in pen

ADG = Average daily gain

WDA= weighted daily average = (weight at 8 weeks-birth weight/days of age)

Dr. Walker also provided a list of contacts for those who performed the analytical work and data analysis. This list can be located at O:\Individl\ONADE\DeLancey\ViaGen\ViaGen Contact List.doc.

Blood, hormone, urine, and meat analysis were performed under the same guidelines in both Experiments 1 and 2.

Blood samples were collected at ages 12 days and 100 days, and on the day of slaughter in experiment 2.

Hormone analysis was run at 12 days and at slaughter.

Urine analysis was performed at 12 days and at slaughter.

Benyshek & Hough Consulting Services are currently performing statistical analysis on this data.

Meat analysis was performed when the animals were slaughtered at the approximate weight of 270 pounds. The tissue samples were frozen prior to analysis. The analyses were performed within a month of slaughter. Carcass data was collected after a 24-hour chill period, and no significant differences were found.

Eurofins is analyzing composition of muscle samples which was expected to be completed by the end of April.

Update: This data is now available at O:\Individl\ONADE\DeLancey\ViaGen\Viagen Report Final 5-17-05.xls

Clarifications Requested

CVM requested information on when the clones from Experiment 1 were transferred from SPF conditions to herd conditions.

ViaGen agreed to provide a timeline of all the clones from Experiment 1.

CVM asked whether it was possible to review the formal veterinary records from Experiments 1 and 2. Dr. Polejaeva stated that Experiment 2 was run by the Clay Center, and was run at the Clay Center’s discretion, and it was unknown whether formal veterinary records existed. Dr. Dubbin explained that veterinary observations may help put other data, such as blood results, in perspective.

Update: The contact for the Clay Center is Ted Atkins at 402-762-4139.

Dr. Dubbin also requested a record of which gilts required assistance in farrowing, and whether these litters were the progeny of boar clones or comparators.

Dr. Polejaeva also stated that more data would be forthcoming regarding meat analysis, proximate information, and quality assurance.

Action Items:

Viagen will contact all of the people involved in the analysis of the samples and data to ensure that FDA staff can contact them directly with any questions that we might have.

Viagen will see if Eurofins will release from their QC group all of the meat composition with the exception of Vitamin B1 which is still being analyzed in the lab.

Mary Bartholomew will contact the consulting statistician to see if he will provide the statistical program that he has written and the data in a form that can be easily read into the statistical program. She is also going to explore the types of statistical analyses that have been performed.

The group discussed that it would be useful to have significance test, outlier test, and mean with some measure of the variation around the mean (standard deviation, etc.) as a basis for generating the same type of figures for the progeny of clones that currently exist in the risk assessment.

Julia Oriani was directed to contact Eurofins by telephone to obtain information on the nutrititional analyses. The phone call should be written up and sent to Larissa.

The CVM group will need to get together soon to make sure that we have the information we need, and that we develop a mutually agreeable work plan. Once that plan has been developed, Larisa asked that as the reviewers conduct their analysis of the data and draw conclusions that they write the information up in a format which is similar to that which is in the current risk assessment.