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Modern Reproductive Biotechnologies and Trade of Germplasm



Professeur Michel THIBIER - France
Present address :
Représentation permanente de la France auprès de l'OAA/FAO
52 Corso del Rinascimento
00186 ROME - Italie

Since the beginning of domestication (a long time ago…), exchange of domesticated animals between neighbors, villages, regions etc… has taken place making it obviously feasible. However in modern ages, there are a number of constraints that have arisen, particularly linked with the sometimes long distances of their transportation. They are of socio-economics nature including the welfare issue but also in relation with the animal health status of those animals. The diseases encountered in one given area may be radically different from those of another and the individuals may therefore carry over pathogens with them. There are means to prevent that but they are usually costly on one hand and sometimes unacceptable due to the high risks that may be associated with such movements on the other hand. The reproductive biotechnologies in relying on gametes or embryos are particularly well adapted to global trade in being associated with a much higher level of safety together with a much-reduced cost. One major technical constraint is the necessity to keep those entities alive during the exchanges. Deep freezing has been on this regard an essential achievement wherever this technique has been effectively developed, because this allows reports both in time and in space. We will see here that deep freezing is not yet applicable for all technologies. It should be stressed however that these most interesting opportunities for international movement do not preclude at all, thorough health surveillance according to relevant risks analysis.

The reproductive biotechnologies include classically (Thibier, 1990) four generations: the first is that of Artificial Insemination, the second, Embryo Transfer, the third comprehends sexing of embryos, in vitro produced embryos and nuclear transfer (cloning). The fourth, the most recent, is that of transgenesis. The present paper aims at reporting on the major features of the risk analysis of carrying pathogens with those gametes or embryos and will show how some of those biotechnologies will contribute most effectively to enhance international movements of germplasms. The bovine species will here be taken as a model.


This biotechnology of the first generation started to be developed on the field just after the last world war more than 50 years ago. Since then, it has a worldwide implementation and more than 100 millions first AI's in the bovine were recorded last year (Thibier and Wagner, 2000). Several million doses of bovine semen and thousands of small ruminants or swine semen are being moved internationally every year. The risk analysis first shows that a significant number of pathogens, viruses but also bacteria, specific or non-specific can enter the male genital tract and be found associated with the semen. The Office International des Epizooties (OIE) distinguishes diseases that are the most at risks (list A of the International Animal Health Code) and those (list B) that although at risks are not quite to the same magnitude a threat to the recipient herd, region or country. Most of the pathogens of the list A diseases may be found in the semen in contaminated donor sires. Among those, are the Foot and Mouth disease virus, the rinderpest virus, the blue tongue virus, contagious bovine pleuro pneumonia mycoplasma etc… Those pathogens if present in semen, could contaminate dramatically not only the female to which the semen is inseminated but also the whole herd or a significant part of a given area. Many of the list B agents are also possibly associated to semen. Hence a thorough management of the donor males has to take place in order to prevent such things to happen. More than 50 years of experience has allowed the industry to fully control the procedure to be put in place. The principle of management is the following: " a donor bull should be free of a given pathogen and included in a collection of individuals also free of this given pathogen". This infers that the male donors should be in a Semen Collection Center (SCC) which is free of those pathogens. In order to do so, bulls have to originate from herds free of the disease, be placed in quarantine stations to monitor them by adequate tests from blood and semen and once in the SCC, with restricted access, again at regular intervals be also monitored. Provided that those conditions are met, then semen can be qualified as "pathogen free" and be safely released for international trade. The health conditions of the environment (region or country) however do remain as a constraint, as in some cases, the individual could be a so-called "healthy carrier" of a pathogen. The full success of deep freezing in most species of farm animals provides an additional advantage.


This second generation was launched on the field by the mid-seventies and consists of collecting embryos at the right stage (7 days after insemination) after in vivo fertilization. Embryos are then collected from a donor and either transferred as fresh to a recipient or deep frozen, stored and transferred in other herds, regions or continents. The number worldwide is less (by 3 logs) than the number of AI's and is in the magnitude of 0.5 million each year (Thibier, 1999). It requires more expertise and investments than AI. However it is also distributed worldwide although North America and Europe represent the two thirds of the total number of embryos transferred. The export of embryos increases every year and many thousands of them are being shipped from one country to another. The risk analysis made by the International Embryo Transfer Society (Import/Export Committee) has shown that pathogens both from the list A and B of the OIE International Animal Health Code could also be associated with embryos collected. The management of such risks is radically different of that in place for AI. The donors, the donor dairy cows for example, cannot easily be assembled together in a given donor center. In addition, originating from various herds with possibly distinct health statuses, those animals would be at risks of becoming affected by a given disease. This among other reasons, explain why the approach taken by the OIE Code relies on the concept of 'officially approved embryo transfer teams" in order to give full guarantee to the importing country that no pathogen is associated with the embryos moved and transferred. This official accreditation relies on four criteria: (1) a vet in the team should be responsible of the operation, (2) the equipment of the team, both in terms of facilities and equipment stricto sensu should be adequate and assessed as such by the official veterinarian authority, (3) the code of practice elaborated by the IETS should be strictly followed by the team and (4) control and regular monitoring should be done by the official veterinarian authorities. A special note should be made in the code of practice of the IETS (written in the IETS Manual, in English, French and Spanish), it includes a thorough washing (10 times etc…) of the embryos which was shown in most instances effective in eliminating the association of a possible pathogen to the embryo. It has been possible to claim that "provided that all those conditions are met, then the embryo transfer is the safest means of moving embryos from one herd to another" (Thibier, 1991). This further allows one to claim that at the present time, any given germplasm can be safely moved to any herd of the planet. The BSE crisis has of course raised new questions. It has been so far shown that the BSE agent was not able to be associated with embryos. However, the large experiment started in the UK in the early nineties is not entirely completed and the final results should be released in the middle of next year (Wrathall, 1997). The absence of case of this disease in a given herd obviously reinforces the level of safety.


This generation includes embryo sexing, in vitro produced embryos and nuclear-transferred embryos (clones) for which the risk analysis gives radically distinct results.

The embryo sexing necessitates the invasion of the zona pellucida (zp), preventing the latter to efficiently exert its protective role as far as the possible pathogens associated to it are concerned. The action to be taken can be easily solved as stated in the OIE International Animal Health Code. Proper management consists in this case to ensure that, at the time of the biopsy, which would, result in invading the zp, the embryo environment, the medium in which it is put, is sterile. This can be achieved by proper washing of the embryo(s).

The in vitro produced embryos are being transferred since a more recent time, the early nineties, and the total number of such embryos in the bovine, recorded worldwide is in the magnitude of 30 000 in 1998 (Thibier, 1999). In addition, technically, even in the bovine, the deep freezing technique is not quite as efficient in terms of pregnancy rates than from fresh embryos. This has considerably slowed down the international movements of such embryos. The female gametes (occytes) used for in vitro fertilization may derive from sampling of the ovaries collected at the abattoirs or from in vivo collection, the so-called ovum pick up technique. The risks in those two situations are quite distinct. In addition, it has been recently shown by several authors that two pathogens studied as a model, namely the IBR/IPV virus (herpes virus) and the BVD virus (pestivirus) could remain associated to the embryos during the procedure. Recently it has also been shown that Foot and Mouth Disease virus could also stick to the embryo in vitro produced. It should be added that the in vitro production of embryos comprehends several steps and may in some cases include a co-culture system for previously in vitro fertilized embryos before they are transferred. This co-culture system adds one significant factor of possible contamination The OIE Code has already taken into consideration those scientific data. Such embryos can however be moved internationally and will be as soon as better deep freezing techniques emerge. Some recent data seem to be promising in this area. The concept relies on a somewhat similar one to the in vivo derived embryo; namely the "official approval of embryo production teams". The criteria to be taken into account are close to those in use for the embryo collection teams and some more monitoring of the immediate environment of the embryos will result also in safe movement of such embryos. There is no doubt that in the future due to its potential, capable of releasing more embryos per given donor, such embryos will be widely moved internationally at a competitive cost.

The nuclear-transferred (cloned) embryos for which an enucleated oocyte is to be associated with a nucleus raises new questions. It should be first recalled that such embryos are produced at this stage almost exclusively for research purposes. However they may serve as an additional tool in the process of generating transgenic animals for duplicating them. The health conditions for such embryos should therefore be visited with great caution. At this time, very little research has been made on their properties to be associated with pathogens. The IETS ad hoc committee, currently studies the possible consequences in terms of diseases that could be so generated, of having a totally new and foreign cytoplasmic environment to the nucleus. It cannot be excluded at this stage that some parts of the genome regulated by the cytoplasmic structure, such as those of pro- or retro-viruses could be then deregulated. So many unknown factors are here to preclude at this time, any free movement of such embryos.


These embryos like those produced by nuclear transfer are either the result of fundamental investigations or of very specific purposes in relation with the biopharming industry. There are so many scientific question marks that it is unreasonable at this time to recommend any international movement of such individuals.

In conclusion, the three first generations that include Artificial Insemination, Embryo Transfer, embryo sexing and in vitro production of embryos are now launched on the field. The two first of those have been used for decades and the deep freezing technique is quite operational. It has allowed an active international movements of germplasm with a high level of safety. They both have comparative advantages to live animals, both of socio-economic nature and healthwise. There is no doubt that as soon as the deep freezing technique of in vitro produced embryos will be under consistent control the latter will provide another alternative to the international movements of germplasm. Those reproductive biotechnologies widely contribute to enhance the international trade, although they sometimes need some specific investment (particularly for the latter generation). In order that these biotechnologies fully play their role as a very safe means of moving genes, a certain number of health constraints and provisions have to be met and always should be so that those reproductive biotechnologies continue to exert their enormous potential in giving access of any desired germplasm to any farmer in the world.

Rome, 25 September 2000.

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