s649

Acta Scientiae Veterinariae. 38(Supl 2): s649-s659, 2010.

ISSN 1678-0345 (Print)
ISSN 1679-9216 (Online)
Biosecurity its added value to Embryo Transfer
Michel Thibier
ABSTRACT
Background: Both in vivo derived (IVD) and in vitro produced (IVP) embryos in cattle have been transferred worldwide
for many decades with great success. Among the many reasons for this to occur, one concerns the Biosecurity
issues which if managed adequately may add a significant value to this Embryo Transfer (ET) technology. There are
a number of conditions for this to be the case.
Review: The present review emphasized the critical points all along the procedures which allow the practitioners to
provide a high degree of safety to their operations because if not under strict care, pathogens mainly from the genital
tract of the donors of embryos or of oocytes but not exclusively, may be associated to the embryos and hence
contribute to spread diseases into the herds of the recipients. Sound scientific data have been elaborated by the
researchers along the last decades which have been able to generate appropriate regulatory measures. Risks at
stakes are here reported, both from in vivo collected or from in vitro produced embryos. They mainly concern various
viruses and bacteria such as the Foot and Mouth disease virus or others and this review summarized the most
relevant studies referring to them. We also described the sequences of events along the procedures that might
contribute to such an association between the pathogens and the embryos. The knowledge of such sequences
allowed the embryo transfer industry to adopt appropriate means to manage the risks and hence to mitigate almost to
a negligible level such risks and hence providing the Biosecurity added value to this technology. Those means have
been identified and assessed through an active participation of the International Embryo Transfer Society/ Health And
Safety Advisory Committee (HASAC), then acting as an ad hoc committee to the World Animal Health Organization
(OIE). The recent 4
th
edition of the IETS Manual (2009) provides an excellent document for the practitioner to proceed
safely. We here summarized some of the most important steps and in particular the way the embryos should be
handled once the in vivo derived embryos or the oocytes for in vitro produced embryos are collected. For the in vitro
produced embryos, emphasis was here given on the collection of ovaries when this is to occur from abattoirs
stressing the point that no animal should be present in the abattoir chain for depopulation reasons when ovaries are
collected for IVP embryos. The close relationship between IETS and OIE has allowed the OIE to introduce those
measures as recommendations into its Terrestrial Animal Health Code giving the international world, efficient and
workable proven guidelines. The present review reported also on the basic principles to follow to give its full degree of
Biosecurity. Those principles rely on the critical and ethical role of the embryo transfer or the embryo production
teams under the leadership of one veterinarian.
Conclusion: several decades of experience with close to 10 million of embryos transferred since the beginning of
this century have demonstrated that providing that those rules are strictly followed, biosecurity is a real added value
to these technologies.
Keywords: cattle, embryo transfer, biosecurity, pathogens, risks assessments, risks management.
Saint Sauveur 64120 Saint Palais France
22_SBTE_THIBIER.P65 4/8/2010, 06:21 649
s650
Thibier M. 2010. Biosecurity its added value to Embryo Transfer.
Acta Scientiae Veterinariae. 38 (Supl 2): s649-s659
I. INTRODUCTION
II. TRANSFER OF IN VIVO DERIVED EMBRYOS
THE RISK ASSESSMENT
THE RISK MANAGEMENT
III. TRANSFER OF IN VITRO PRODUCED EMBRYOS
THE RISK ASSESSMENT
THE RISK MANAGEMENT
IV. CONCLUSIONS
I. INTRODUCTION
The second and third generations of the Reproductive Biotechnologies, respectively embryo transfers of in
vivo derived embryos and of in vitro produced embryos have now been used for more than a third of a century for the
former and a quarter of a century for the latter, in other words for quite a significant period of time. Moreover the latest
report of the International Embryo Transfer Society Data Retrieval Committee [19] in cattle and in 2008, shows that
800 000 embryos were transferred worldwide (two thirds as in vivo derived embryos and one third as in vitro produced)
and from almost all continents and regions. Those figures have increased more or less gradually along the last years
but have remained in the same order of magnitude for the last 10 years or so which results in the fact that for the first
decade of the 21
st
century, roughly close to 10 millions of bovine embryos have been so transferred from one herd to
another wherever the recipient herd may be located, in the same farm as the donor animals or to different farms,
towns, provinces, countries, regions, or continents And yet no major contamination of pathogens associated with
the embryos proper has ever been identified. Clearly there have been some major crisis in the disease epidemiology
of livestock those last two decades or so in many parts of the world but never have those diseases occurrences been
associated with the transfer of embryos.
So, those two Reproductive Biotechnologies do have a significant added value to their own potential of
transferring germplasm from one donor animal to a recipient and this is to provide full biosecurity in such operations.
This advantage does not imply at all that there are no risks at stakes but only results from appropriate measures
implemented to manage such risks, measures that have been identified, tested and validated.
The aims of the present review are in taking the bovine species as a model, first to recall the risks at stakes
and second to report on and describe those measures, constraints and critical points to follow by the practitioners
which only can assure this potential of added value of Biosecurity to embryo transfers.
II. TRANSFER OF IN VIVO DERIVED EMBRYOS
The risk assessment
In order to implement with efficiency appropriate measures to ensure that transfer of in vivo collected
embryos exerts its full impact in terms of biosecurity, it is necessary to clearly identify the risks that are at stakes
when collecting, handling and transferring such embryos.
Pathogens can be shed in the genital tract and hence may be one first source of contamination to the
embryo to be collected if present at the time of collection or between fertilization and collection. So, as soon as these
in vivo derived embryo collections and transfers started to be implemented and thanks to the veterinary community,
the latter started to investigate the risks and by the same token tested appropriate means to mitigate such risks. For
example, it was interesting to see published in 1979, a paper by Wrathall and Mengeling [28] showing in the pig for the
first time very demonstratively, the risk of contaminating the recipients with infected embryos. So there are such risks
22_SBTE_THIBIER.P65 4/8/2010, 06:21 650
Thibier M. 2010. Biosecurity its added value to Embryo Transfer.
Acta Scientiae Veterinariae. 38 (Supl 2): s649-s659
s651
and many investigations have been performed those last 30 years, on the interaction between pathogens and embryos
and a complete set of references (more than 400) can be consulted on the International Embryo Transfer Society
(IETS) web site [www.iets.org]. For example in the bovine, 89 species of pathogens have been investigated, some of
them being considered as the most dangerous, classified in the former category A diseases of the OIE, such as the
Foot and Mouth Disease (FMD), Rinderpest, Blue tongue (BT), Contagious Bovine Pleuro Pneumonia (CBPP). Also
were investigated diseases that are economically devastating in some circumstances, such as the Infectious Bovine
Rhinotracheitis, Infectious Pustular Vaginitis (IBR/IPV) disease due to the Bovine Herpes virus 1 (BHV1) or else the
Bovine Viral Diarrhea disease (BVD). Table 1 summarizes some of the data collated in the IETS Manual [12]. These
results show that for most diseases and provided that defined sanitary practices (see below) are followed during the
process, the risks of transmission of a given disease from the donor to the recipient via an embryo is minimum.
However in some circumstances and for a few diseases, added measures such as those recommended in the IETS
Manual (trypsinization for example) have to be implemented (see below).
The mechanism of such associations has been recently reviewed by Van Soom et al. [25] in the IETS
latest edition of the Manual, showing the key factor of the zona pellucida (ZP) in such interactions.
The risks to have a pathogen associated with a given embryo result from a sequence of events that has
been nicely summarized from an epidemiological standpoint by Stringfellow and Givens [13]. Such a sequence
includes 1) the exposure to pathogen, 2) the continued association of pathogen with the embryos, 3) the maintenance
of infectivity of pathogen throughout embryo manipulation and processing and finally 4) delivery of an infective dose
of pathogen to a susceptible recipient. All those steps will be the targets of the measures to be implemented in order
to provide all the biosecurity to this technology.
But to this sequence, there is the whole process of manipulating and storing the embryos that could carry
over some hazards and hence which should also be under strict control. A special attention was given recently by
Wrathall et al. [29] to the risk of embryos being fertilized with pathogen- infected semen. From their studies, these
authors concluded for in vivo derived embryos, the risk of transmitting the disease when semen infected with
Enzootic Bovine Leukosis virus (EBLV) or Blue Tongue Virus (BTV) is used for AI or natural service of the embryo
donors, is negligible and the same is almost certainly true for semen infected with Bovine Herpes Virus 1 (BHV1)
if the embryos are also treated with trypsin. There were however some reservations for BVDV, although from field
studies so far suggests that this is very unlikely.
Table 1. Summarized results of studies of pathogens intact zona pellucida embryos interaction (derived from
the International Embryo Transfer Society Manual, 3rd edn, 1998) [12].
Types of pathogens No. embryos exposed (a) Assay of embryos
In vitro contamination and assay of bovine embryos (b)
Viruses 12169 0
Other viruses(c) 29144 36 100% positive
Bacteria 3896 026% positive
Mycoplasmas 20111 30100% positive
Assay of embryos from zona pellucida intact bovine embryos from infected or seropositive donors
Virus 2372(d) Negative
Brucella 309 Negative
Chlamydia 5 Negative
(a) Range of number of embryos per pathogen studied.
(b) High concentration exposure mimicking a worse case scenario.
(c) Bovine herpes virus 1, bovine herpes virus 4 and Vesicular Stomatitis Virus (VSV).
(d) Foot and mouth disease virus-infected donors.
22_SBTE_THIBIER.P65 4/8/2010, 06:21 651
s652
Thibier M. 2010. Biosecurity its added value to Embryo Transfer.
Acta Scientiae Veterinariae. 38 (Supl 2): s649-s659
A special notice should be given to the risks associated with materials of animal origin. Any biological
product of this kind used for recovery of gametes, sperm and oocytes or embryos, dilution, in vitro maturation of
oocytes, washing and storage is potentially a source of contamination. This is of particular relevance with regard to
the Transmissible Spongiform Encephalopathies (TSE) as discussed at large by Wrathall [27].
The putative contamination of semen or embryos while stored in liquid nitrogen (LN) tanks for example, as
an additional source of contamination, has also received recent attention. Bielanski et al. [4] have demonstrated the
occurrence of microflora in LN tanks such as Stenotrophomonas maltophilia that was able in experimental contact
with semen, to decrease the motility of this semen. These authors have indicated that direct contact of contaminated
LN with embryos may lead to their association with viral agent. However, they have also shown that all sealed
samples of embryos stored in contaminated LN tanks tested negative for the presence of bacteria or viruses. Similarly,
this author [1] showed that the vapor phase of liquid nitrogen is a safe means for short-term storage and transportation
of embryos in so-called dry shipper dewars. A recent and comprehensive review on the risk of contamination of
germplasm during cryopreservation and cryobanking [2] has been published in the latest edition of the IETS Manual.
For in vivo-derived embryos, and with the proviso that all guidelines published by IETS and OIE after
official approval, are rigorously followed (see below), the IETS/ HASAC [Health And Safety Advisory Committee]
relevant committee, has categorized diseases according to the risks assessment analysis, into four categories. The
category one is that for which sufficient data are available to determine the risks to be negligible provided that the
embryos are properly handled between collection and transfer. As seen in the table 2, there are only eight diseases
listed in this category and it is unlikely, unfortunately, that this number will increase in the near future due to the
insufficiency of research in this area.
Table 2. List of International Embryo Transfer Society (IETS)/World Animal Health Organization (OIE) diseases
in category 1 [26].
Disease Species Note
Foot and mouth disease Cattle
Enzootic bovine leucosis Cattle
Blue tongue Cattle
Brucella abortus Cattle
Infectious bovine rhinotracheitis Cattle Trypsin treatment required
Pseudorabies Swine Trypsin treatment required
Bovine spongiform encephalopathies Cattle
Scrapie Sheep
Scrapie in sheep has been added very recently according to the conclusions of the Research sub-Committee
of the IETS/Health and Safety Advisory Committee (meeting of 2010, in Cordoba, Argentina) and in the process of
being approved by the World Animal Health Organization (OIE) in 2010 [OIE, Terrestrial Animal Health Code, chapter
4.7].
The risk management
Those procedures to follow in order to assure full safety to the herds in which recipients are to receive in
vivo collected and transferred embryos are described in details in the latest edition of the IETS Manual [14]. It is a
code of good practice and should be included in a quality assurance system wherever possible.
The first critical step of course refers to the thorough clinical examination of the donor animal and its
environment (lack of infectious contagious disease in the area or in the herd). As pointed out by Wrathall et al. [29], the
semen used for inseminating the donors should not be forgotten in the check list. It is always beneficial in terms of
risks management and/or compulsory, particularly in the context of international exchanges, to use semen processed
from semen collection centres that are officially accredited (see OIE, 2010 Animal Terrestrial Code, chapters 4.5 and
22_SBTE_THIBIER.P65 4/8/2010, 06:21 652
Thibier M. 2010. Biosecurity its added value to Embryo Transfer.
Acta Scientiae Veterinariae. 38 (Supl 2): s649-s659
s653
4.6) [26] because of being under official supervision.
For the handling of embryos, the basic recommendations widely described by Stringfellow [11] in the latest
edition of the IETS Manual, can be summarized as follows. The first stage is to ensure an appropriate washing, 10
times consecutively with a new pipette each time, with immersion of the embryo(s) in each wash for duration of 1 min.
with light agitation and with at least a dilution factor of 1/100 between each washing. There are now means to do this
in a convenient manner and consuming little time. The embryo should be very carefully inspected under magnification
(X 50) and should only been processed if the embryo has an intact zona pellucida and no adherent debris because
such cells could serve as a source of contamination and allow for carry over the pathogen. The treatment of embryos
with the enzyme trypsin is often recommended when dealing with sticky pathogens such as the herpes virus BHV1.
This was shown not to be always necessary [20] but is nevertheless a good procedure and often required for exported
embryos. The way trypsin is to be handled is also relevant since as a protein enzyme, it is quite sensitive to the
environment. It should also be mentioned that such a treatment is not by any mean, a panacea. Even if used, it should
not be considered as replacing the need for sanitary precautions with the environment of the embryos.
The media may also be of some concern as discussed above. Its nature and origin should hence be
selected with great care. The addition of antibiotics is also of some value if used appropriately.
The quality control of the whole process is now necessary for a given team and regular testing in the media
collected and stored for assay should be a standard procedure. This could involve search for a putative contamination
by various viruses that might originate from the collected donor of from some serum used in the media, and the status
for pathogenic and also for saprophytic microflora. This should contribute in the mid-term to establish and verify the
effectiveness of the quality assured production process procedure.
These procedural considerations are part of the OIE recommendations (World Animal Health Organization,
Terrestrial Animal Health Code 2010, chapter 4.7.) [26] that specifically refer to the guidelines published in the IETS
Manual. They are also most of the time included in the regulations for moving embryos from one farm to another.
In doing so, it is right to state that embryo transfer contributes to improving the animal health status of a
given population in controlling very strictly such movements of germplasm between herds.
The basic concept of those regulations relies on that of the official approval of embryo transfer teams. This
was a very important step in the scope of the veterinary regulations that generally rely on the animals, its confinement
and its products. Here the safety of the industry fully relies on the ethical and technical excellence of the man/woman
in charge, head of the embryo transfer team [18].
The criteria used by the veterinary authorities to give their official approval rely on the relevant chapter
(chapter 4.7) of the OIE Code. According to it, the embryo collection team is a group of competent technicians,
including at least one veterinarian, to perform the collection, processing and storage of embryos. The following
conditions should apply:
1. The team should be approved by the Competent authority,
2. The team should be supervised by a team veterinarian.
3. The team veterinarian is responsible for all team operations which include verification of donor health status,
sanitary handling and surgery of donors and disinfection and hygienic procedures.
4. Team personnel should be adequately trained in the techniques and principles of disease control. High
standards of hygiene should be practiced to preclude the introduction of infection.
5. The collection team should have adequate facilities and equipment for:
a. collecting embryos;
b. processing and treatment of embryos at a permanent site or mobile laboratory;
c. storing embryos.
These facilities need not necessarily be at the same location.
6. The embryo collection team should keep a record of its activities, which should be maintained for inspection by
the Veterinary Authority for a period of at least 2 years after the embryos have been exported.
7. The embryo collection team should be subjected to regular inspection at least once a year by an Official
Veterinarian to ensure compliance with procedures for the sanitary collection, processing and storage of embryos.
22_SBTE_THIBIER.P65 4/8/2010, 06:21 653
s654
Thibier M. 2010. Biosecurity its added value to Embryo Transfer.
Acta Scientiae Veterinariae. 38 (Supl 2): s649-s659
The following articles of this chapter deal with the conditions applicable to 1) processing laboratories, 2)
introduction of donor animals, 3) the risk management, 4) collection and storage of embryos, 5) optional tests ands
treatments, 6) storage and transport of embryos and 7) procedure for micromanipulation.
These articles refer, as always from the OIE, to international movements but it is of interest to note the
wording regarding the optional tests.
The testing of samples can be requested by an importing country to confirm the absence of pathogenic
organisms that may be transmitted via in vivo derived embryos, or to help assess whether the degree of quality
control of the collection team (with regard to adherence to procedures as described in the IETS Manual) is at an
acceptable level. Samples may include:
Non-viable embryos/oocytes
Embryo collection (flushing) fluids
Washing fluids, the last four washes of the embryos/oocytes should be pooled (ref. IETS Manual).
Samples, the samples referred to above should be stored at 4C and tested within 24 hours. If this is not
possible, then samples should be stored frozen at -70C or lower.
In conclusion to this part and as far as biosecurity is concerned in the context of in vivo derived embryos,
the system in place worldwide and as approved by OIE has proven to be effective. It is based on science and integrity
in the collection and processing procedures and so provides an immense comparative advantage to this technique in
moving germplasm from one herd to another.
III. TRANSFER OF IN VITRO PRODUCED EMBRYOS
The risk assessment
As already stated [6,17] when dealing with the pathogen-embryo interaction, one should never extrapolate
from one species to another and from one pathogen to another, even if generically very close. This holds certainly true
for the mode of production of embryos: in vivo derived vs. in vitro produced. Due to apparent morphologic differences
in the zonae pellucidae of these two classes [23], some pathogens seem to adhere more readily to the ZP of the IVF
embryos [15,24] and such interactions might differ from one type or subtype of pathogen to another not only between
in vivo derived and in vitro produced embryos but even within in vitro produced embryos. An example of differences
between in vivo derived and in vitro produced is that of FMD virus as shown in table 3.
Table 3. Different interaction of FMD virus (Type 0) with in vivo derived embryos or in vitro produced embryos
(from ref.[17]).
Embryos In vivo derived (*) In vitro produced (* *)
Number 169 73
Type of virus 0
1
0
1
Viral concentration 10
6
pfu 10
7
TCIDSO/ml
Time of exposure 4-18h 4h
Test Plaque and inoculation of epithelium Plaque and PCR
Results Negative Virus isolation in all first
fluids and
cytopathogenically
positive from the
developed and degenerated
embryos
(*) adapted from Singh et al. [10], (* *) adapted from Marquant Le Guienne et al. [8].
22_SBTE_THIBIER.P65 4/8/2010, 06:21 654
Thibier M. 2010. Biosecurity its added value to Embryo Transfer.
Acta Scientiae Veterinariae. 38 (Supl 2): s649-s659
s655
Regarding the subtlety of the association of subtypes of viruses with in vitro produced embryos, a very
interesting experiment was performed by Bielanski et al. [5] dealing with two non cytopathic (NCP) BVDV biotypes,
type 1 (NY-1) or type 2 (PA-131). Those two viruses were experimentally added to bovine in vitro produced embryos.
Then the embryos were treated according to the IETS protocol and transferred to recipients. Part of the results of this
experiment is reported on table 4.
Table 4. Different interaction of subtypes of BVDV with in vitro produced embryos (adapted from ref. [5]).
Type of NCP BVDV NY - 1 PA 131
No Pregnancies /No of transfers 20/33 25/61
Percentage of seroconversions in recipients 0% 51.4%
No of seroconversions in offspring 0 (18 full term calves) 0 (only 2 went to full term
and gave birth)
Virus isolation tests on non transferred embryos (in %) 25% 28%
The authors concluded that a large proportion of recipients that received embryos exposed to BVDV,
especially those exposed to a high concentration of type 2 virus, became infected after ET and their pregnancies
failed. However term pregnancies resulted in calves free of both virus and antibody.
This emphasizes two points: the first is that the whole procedure seems to be quite safe in producing
embryos even in such a worst case scenario, in resulting in non contaminated embryos but second, the possibility
of contaminating the recipients and hence the herd which means that regarding potentially BVDV- infected donors or
from batches of ovaries collected at abattoirs, additional biosecurity measures have to be taken.
As proposed by Thibier and Gurin [22], the sequence of hazards in terms of infectious agents includes 1)
those related to the female donor and the mode of collection (abattoir collection or ovum pick up), 2) the maturation
process, 3) the fertilization (introduction of semen), 4) the co-culture in vitro development, 5) the cryopreservation
before 6) the last step, thawing and transfer.
The first point of interaction is the oocyte itself and its follicular environment, (surrounding cells of the
oocytes and the follicular fluid). The magnitude of this risk itself may be modulated according to the source of ovaries
that are being used: either from ovum pick up in which case the donor animal health status may be well identified or
from the abattoir in which case a different approach for risk management has to be taken (see below).
Because contamination of such cells by two types of viruses, BVDV and BHV-1 (IBR/IPV) is not uncommon
(table 5), those two viruses has been extensively investigated by several investigators in different parts of the world
[15].
Table 5. Level of contamination reported in abattoir-origin materials of animal origin used in multiple in vitro
fertilization laboratories (according to ref. [15]).
Contaminant Range of positive samples (%)
Bovine herpesvirus-1 012
Bovine viral diarrhoea virus 112
Bacteria 1368
Collected by the authors from data from Avery et al. (1993), Bielanski et al. (1993), Bielanskiand Stewart (1996), Marquant-Le
Guienne et al. (2000) and Galik et al. (2002), cited in [15].
22_SBTE_THIBIER.P65 4/8/2010, 06:21 655
s656
Thibier M. 2010. Biosecurity its added value to Embryo Transfer.
Acta Scientiae Veterinariae. 38 (Supl 2): s649-s659
Those viruses appeared to adhere to the oocyte zona pellucida and hence are external to the oocyte. This
hazard is complicated by the fact that those viruses often result in an asymptomatic disease and this explains why
particular attention should be given to those pathogens.
As far of the methods of collection are concerned, the risks arc diverse according to that used. In the case
of abattoir collections, the problem is first to ensure that the health status of the lot of females from which ovaries
have been collected are free from infectious or contagious diseases which implies a good tracing system of the origin
of the females. A second level of risk of abattoir collections relates to possible environmental contamination of the
collected material. In the case of ovum pick up, the risks are very much the same as for collecting embryos as
regards the donor female. In addition, there is another source of contamination from the equipment particularly when
a series of animals (which is usually the case) are collected at the same session. Transportation of this material to the
laboratory is another source of external contamination.
An additional source of risks coming from individual animals is that of semen. As indicated above, this
point has been recently revisited [29] and should not be overlooked.
During the handling and the processing of embryos in the laboratory, from collection to transfer, there are
many risks of environmental contamination that need to be controlled (see below). As seen for in vivo derived
embryos, the media are also an important possible source of contamination and moreover, several types of media
may be used during the whole week thereby increasing the risks. A number of media contain products which are of
animal origin. It is strongly recommended to replace wherever possible such products by others such as amino acids
from plant origin. Finally, the contamination of co-culture cells is also at risks, particularly when of primary origin.
Several investigators have reported such contamination by bacteria or viruses such as BVDV or BHV-1 V [7]. The use
of controlled cell lines which have been determined to be pathogen free is recommended whenever possible. Another
approach relies on the use of totally synthetic medium (SOF) which also contributes to lower the risks of adverse
consequences associated with pathogen contamination.
One additional point may be here noted referring to the effect of cryopreservation. The objective a recent
study by Bielanski & Lalonde [3] was to determine the effect of cryopreservation by conventional slow controlled
cooling and by vitrification on the presence of BVDV and BHV-1 infectivity associated with frozen-thawed day 7
bovine IVF produced embryos. Their conclusion was that cryopreservation reduced the proportion of infected embryos
but did not render all of them free from infectious pathogens.
The risk management
A set of recommendations to control risks associated with such embryos have been elaborated within the
IETS and been published in the relevant chapter of the latest edition of the IETS Procedures Manual [9]. Here too,
they should be considered by all practitioners as a mandatory code of good practice.
The first step to survey is the health status of the area, the herd of origin when relevant and the donor
herself making sure that no infectious, contagious disease are present at the time of collecting the oocytes. A special
note is to be given when dealing with animal from a given species or breeds threatened by extinction. It may well be
for reasons of biodiversity or germplasm conservation that the general conditions required are not met. There could
consequently be some exceptions, because of the considerable power of this technique for quality control (see
below) and this, incidentally, constitutes one comparative advantage to this technique. When ovaries are collected
from the slaughter house, it is of the greatest importance to trace back the herd situation of those females and check
for example that they do not come from any depopulated herd for health reasons.
The premises and working areas should be so designed that individual specialized units are set aside for
particular tasks with restricted access. Wherever possible, a laminar flow chamber should be in place with close
attention to cleaning and disinfecting procedures as rightly stated by Gurin et al. [7].
The handling of embryos during the various steps should always be conducted with great care and under
highest hygienic conditions. As stated above, the semen used should be specific pathogen free and it is desirable to
test each lot in IVF before it is used routinely, because some semen with low levels of bacterial contamination has
been problematic according to Stringfellow et al. [15]. The quality of the media and of the co-culture cells system
when relevant is one of the most critical points of the procedure. All biological products should be strictly controlled
and guaranteed free from microorganisms (virus, bacteria or fungi). Sera containing antibodies against agents of
22_SBTE_THIBIER.P65 4/8/2010, 06:21 656
Thibier M. 2010. Biosecurity its added value to Embryo Transfer.
Acta Scientiae Veterinariae. 38 (Supl 2): s649-s659
s657
particular concern should be avoided. It is also strongly advised to have knowledge and confirmation of the inactivation
procedures from the manufacturers when relevant.
Adding antibiotics to the media is also always of good practice as it contributes to remove permanent or
opportunistic pathogenic agents or saprophytic microorganisms inadvertently introduced at the collection point or at
the time of fertilization from semen that can never be sterile [7]. Unfortunately, approved antiviral compounds are not
currently available yet for use in embryo production however promising it might be [Givens, personal communication,
2010].
Finally, the recommended washing procedure such as that described above for the in vivo derived embryos
contributes to further reduce the likelihood of associating pathogens with the embryos so produced and released from
the lab for transfer. One of the major comparative advantages of this technology is that the production system
provides control points and sufficient time to allow for each batch of embryo produced to be monitored and assessed
to relative to their sanitary status. In addition, the many different media used provides an excellent source of sampling
as it has been shown that the media, as a mediate environment of the embryos, serves as a good indicator of the
pathogens to which they could have been exposed during the process [21]. The quality control is here of particular
relevance. Such a control starts with the strict recording in the laboratory book of all the events, from the identification
of the ovaries to the release of the so produced embryos. It should in a routine operation, as indeed is often the case,
include regular sampling of all the media used in the process and any degenerated embryos which give a very
accurate indicator of the environment to which viable embryos may have been exposed. Such tests are also sometimes
required in special circumstances by the veterinary authorities before exports of such in vitro produced embryos, for
example. Here too, the whole system could facilitate the establishment a quality assured production process.
Just like for in vivo derived embryos, these procedural considerations are part of the OIE recommendations
(World Animal Health Organization, Terrestrial Animal Health Code 2010, chapter 4.8.) [26] that specifically refer to
the guidelines published in the IETS Manual. They are also most of the time included in the domestic regulations for
moving embryos from one farm to another.
The basic concept of those regulations relies on that of the official approval of embryo production teams.
Like for in vivo derived embryos and again, this was a very important step in the scope of the veterinary regulations
that generally rely on the animals, its confinement and its products. Here again, the safety of the industry fully relies
on the ethical and technical excellence of the man/woman in charge, head of the embryo transfer team.
The criteria used by the veterinary authorities to give their official approval rely on the relevant chapter of
the OIE Animal Terrestrial animal health code [chapter 4.8. http://www.oie.int/eng/normes/mcode/en_chapitre_1.4.8.htm].
According to this OIE Code, the embryo production team is a group of competent technicians, including at least one
veterinarian, to perform the collection and processing of ovaries/oocytes and the production and storage of in vitro
produced embryos. The following conditions should apply:
1. The team should be approved by the competent authority
2. The team should be supervised by a team veterinarian.
3. The team veterinarian is responsible for all team operations which include the hygienic collection of ovaries
and oocytes and all other procedures involved in the production of embryos intended for international
movement.
4. Team personnel should be adequately trained in the techniques and principles of disease control. High
standards of hygiene should be practised to preclude the introduction of infection.
5. The production team should have adequate facilities and equipment for:
a. collecting ovaries and/or oocytes;
b. processing of oocytes and production of embryos at a permanent site or mobile laboratory;
c. storing oocytes and/or embryos.
These facilities need not necessarily be at the same location.
6. The embryo production team should keep a record of its activities, which should be maintained for inspection
by the Veterinary Authority for a period of at least 2 years after the embryos have been exported.
7. The embryo production team should be subjected to regular inspection at least once a year by an Official
Veterinarian to ensure compliance with procedures for the sanitary collection and processing of oocytes and
the production and storage of embryos.
22_SBTE_THIBIER.P65 4/8/2010, 06:21 657
s658
Thibier M. 2010. Biosecurity its added value to Embryo Transfer.
Acta Scientiae Veterinariae. 38 (Supl 2): s649-s659
The following articles of this chapter deal with the conditions applicable to 1) processing laboratories, 2)
donor animals, 3) optional tests and treatments, 4) the risk management, 5) storage and transport of embryos and 6)
procedure for micromanipulation.
The article 4.8.4., relevant to the donor animals distinguishes clearly the cases of recovering oocytes from
live donors from those from batches of ovaries collected from an abattoir. For the latter, the abattoir should be officially
approved and the animals should not have been designated for compulsory slaughter for a notifiable disease.
In conclusion to this part, it is clear that the processing procedures of IVF embryo production require
particular care from the embryo production team because a few pathogens have been proved to adhere more readily
to the zona pellucida of such embryos. However the whole procedure processed in a well established laboratory with
competent personal under the leadership of the team veterinarian gives appropriate means to safely release and
transfer such IVF embryos. So, this generation of Reproductive Biotechnologies can also, like for in vivo derived
embryos, give all guarantee of a high level of biosecurity.
IV. CONCLUSIONS
In conclusion, the statement made some 21 years ago [16] that transfer of in vivo derived embryos was the
safest mean of exchanging genes between herds, areas or continents remains more than valid. Nothing during those
years contradicted this statement thanks to the high degree of professionalism and good practice of the embryo
transfer industry based on sound research that has been done and passed over to the regulatory agencies. Regarding
the second type of embryos, i.e. those produced by in vitro fertilization and culture, recent research, particularly on
BVDV has confirmed that some types or subtypes of viruses could be strongly associated with the zona pellucida of
such IVP embryos. This gives even more responsibility to the embryo production team to strictly follow its commitment
to their official approval by the competent veterinarian authority. There are rules to be followed that have shown that
under such conditions, this technology too could give full guarantee in terms of biosecurity. Such possibilities at the
time of world globalization with increasing international trade, provides to those technologies a clear added value.
REFERENCES
1 Bielanski A. 2005. Non-transmission of bacterial and viral microbes to embryos and semen stored in the vapour phase of liquid nitrogen
in dry shippers. Cryobiology. 50: 206-210.
2 Bielanski A. 2009. Risk of contamination of germplasm during cryopreservation and cryobanking. Appendix C. In: International Embryo
Transfer Manual. A procedural guide and general information for the use of embryo transfer technology emphasizing sanitary procedures.
4
th
Edition. (IETS Publish., Savoy, USA). 131-139.
3 Bielanski A. & Lalonde A. 2009. Effect of cryopreservation by slow cooling and vitrification on viral contamination of IVF embryos
experimentally exposed to bovine viral diarrhea virus and bovine herpesvirus-1. Theriogenology. 72: 919-925.
4 Bielanski A., Bergeron H., Lau P. C. K. & Devenish J. 2003. Microbial contamination of embryos and semen during long term banking
in liquid nitrogen. Cryobiology. 46: 146-152.
5 Bielanski A., Algire J., Lalonde A. & Nadin-Davis S. 2009. Transmission of bovine viral diarrhea virus (BVDV) via in vitro-fertilized
embryos to recipients, but not to their offspring. Theriogenology. 71: 499-508.
6 Givens M.D., Gard J.A. & Stringfellow D.A. 2007. Relative risks and approaches to biosecurity in the use of embryo technologies in
livestock. Theriogenology. 68: 98-307.
7 Gurin B., LeGuienne B. & Thibier M. 2000. A secure health status associated with the production and trade of in vitro derived cattle
embryos. Livestock Production Science. 62: 271-285.
8 Marquant-LeGuienne B., Rmond M., Cosquer R., Humblot P., Kaiser C., Lebreton C., Crucire C., Gurin B., Laporte J. &
Thibier M. 1998. Exposure of in vitro produced bovine embryos to foot and mouth disease virus. Theriogenology. 42: 579-590.
9 Marquant-LeGuienne B., Guyader-Joly C., Ponsart C. & Gurin B. 2009. General sanitary procedures associated with in vitro
production of embryos. In: International Embryo Transfer Manual. A procedural guide and general information for the use of embryo
transfer technology emphasizing sanitary procedures. 4
th
Edition. (IETS Publish., Savoy, USA). 57-64.
10 Singh E.L., McVicar J.W., Hare W.C.D. & Mebus C.A. 1986. Embryo transfer as a means of controlling the transmission of viral
infections. VI. The in vitro exposure of bovine and porcine embryos to foot and mouth disease virus. Theriogenology. 29: 587- 593.
11 Stringfellow D.A. 2009. Recommendations for the sanitary handling of in vivo derived embryos. In: International Embryo Transfer
Manual. A procedural guide and general information for the use of embryo transfer technology emphasizing sanitary procedures. 4
th
Edition. (IETS Publish., Savoy, USA). 65-68.
12 Stringfellow D.A. & Seidel S.M. 1998. International Embryo Transfer Manual. A procedural guide and general information for the use of
embryo transfer technology emphasizing sanitary procedures. 3
rd
Edition. (IETS Publish., Savoy, USA). 172 pp.
22_SBTE_THIBIER.P65 4/8/2010, 06:21 658
Thibier M. 2010. Biosecurity its added value to Embryo Transfer.
Acta Scientiae Veterinariae. 38 (Supl 2): s649-s659
s659
13 Stringfellow D.A. & Givens M.D. 2000. Preventing disease transmission through the transfer of in vivo derived bovine embryo.
Livestock Production Science. 62: 237-251.
14 Stringfellow D.A. & Givens M.D. 2009. International Embryo Transfer Manual. A procedural guide and general information for the use
of embryo transfer technology emphasizing sanitary procedures. 4
th
Edition. (IETS Publish., Savoy, USA). 151 pp.
15 Stringfellow D.A., Givens M.D. & Waldrop J.G. 2004. Biosecurity issues associated with current and emerging technologies.
Reproduction, Fertility and Development. 16: 1-10.
16 Thibier M. 1989. Le transfert embryonnaire: le moyen le plus sr, au plan sanitaire dchange de gnes. In: the Proceedings of Sixth
Scientific meeting of the AETE. Lyon (France). 67-81.
17 Thibier M. 2001. Identified and unidentified challenges for reproductive biotechnologies regarding infectious diseases in animal and public
health. Theriogenology. 56: 1465- 1481.
18 Thibier M. 2006. Biosecurity and the various types of embryos transferred. Reproduction in Domestic Animals. 41: 260-267.
19 Thibier M. 2009. The worldwide statistics of embryo transfers in farm animals. International Embryo Transfer Society Newsletter. 27(4):
13-19.
20 Thibier M, & Nibart M. 1987. Disease control and embryo imports. Theriogenology. 27: 37-47.
21 Thibier M. & Gurin B. 1993. La scurit sanitaire dans le transfert dembryons bovins collects in vivo, fconds in vitro ou clons.
Bulletin Acadmie Vetrinaire de France. 70: 183-184
22 Thibier M. & Gurin B. 2000. Embryo transfer in small ruminants: the method of choice for health control in germplasm exchanges.
Livestock Production Science. 62: 253-270
23 VanRoose G., Nauwynck H., Van Soom A., Ysebaert M.T., Charlier G., Van Oostvelt P. & de Kruif A. 2000. Structural aspects of the
zona pellucida of in vitro-produced bovine embryos: a scanning electron and confocal laser scanning microscopic study. Biology of
Reproduction. 62: 463-469.
24 Van Soom A. & Nauwynck H. 2008. Risk associated with bovine embryo transfer and their containment. CAB reviews: Perspectives in
Agriculture, Veterinary Science, Nutrition and Natural Resources, 7pp. htpp://www.cababstractsplus.org/cabreviews.
25 Van Soom A., Nauwynck H. & Wrathall A.E. 2009. Scientific foundations for the epidemiological safety of embryo transfer. In:
International Embryo Transfer Manual. A procedural guide and general information for the use of embryo transfer technology emphasizing
sanitary procedures. 4
th
Edition. (IETS Publish., Savoy, USA). 13-40.
26 World Animal Health Organization (OIE). 2010. Terrestrial Animal Health Code. Chapters 4.5 4.8. (http://www.oie.int/eng/normes/
mcode/en_sommaire.htm).
27 Wrathall A.E. 2000. Risks of transmission of spongiform encephalopathies by reproductive technologies in domestic ruminants.
Livestock Production Science. 62: 287-316.
28 Wrathall A.E. & Mengeling W.L. 1979. Effect of transferring, parvovirus-infected fertilized pig eggs into seronegative gilts. British
Veterinary Journal. 135: 255-261.
29 Wrathall A.E., Simmons H.A. & Van Soom A. 2006. Evaluation of risks of viral transmission to recipients of bovine embryos arising from
fertilization with virus- infected semen. Theriogenology. 65: 247-274.
www.ufrgs.br/favet/revista
Supl 1
22_SBTE_THIBIER.P65 4/8/2010, 06:21 659
22_SBTE_THIBIER.P65 4/8/2010, 06:21 660