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Thibier M. 2010. Biosecurity its added value to Embryo Transfer.Acta Scientiae Veterinariae.38 (Supl 2): s649-s659

I. INTRODUCTION

II. TRANSFER OF IN VIVODERIVED EMBRYOS

THE RISK ASSESSMENT

THE RISK MANAGEMENTIII. TRANSFER OF IN VITROPRODUCED 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

vivoderived embryos and of in vitroproduced 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 vivoderived embryos and one third as in vitroproduced)

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 21stcentury, 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 epidemiologyof 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 VIVODERIVED EMBRYOS

The risk assessment

In order to implement with efficiency appropriate measures to ensure that transfer of in vivocollected

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 vivoderived 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

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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 vitromaturation 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 theembryos 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 ofbeing 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

vivocollected 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

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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 confinementand 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. Highstandards 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 bythe 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.

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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 vivoderived 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 notpossible, 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 vivoderived 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 VITROPRODUCED 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 vivoderived vs.in vitroproduced. 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 vivoderived and in vitro produced embryos but even within in vitroproduced embryos. An example of differences

between in vivoderived and invitroproduced is that of FMD virus as shown in table 3.

Table 3. Different interaction of FMD virus (Type 0) with in vivoderived embryos or in vitroproduced embryos

(from ref.[17]).

Embryos In vivoderived (*) In vitroproduced (* *)

Number 169 73

Type of virus 01

01

Viral concentration 106pfu 107TCIDSO/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

positivefrom the

developed and degenerated

embryos

(*) adapted from Singh et al. [10], (* *) adapted from Marquant Le Guienne et al. [8].

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Regarding the subtlety of the association of subtypes of viruses with in vitroproduced 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 vitroproduced 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 vitroproduced 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 vitrodevelopment, 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].

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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 vivoderived

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 collectedfrom 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

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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 vivoderived 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 veryaccurate 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 vivoderived 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 vivoderived 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 ovariesand oocytes and all other procedures involved in the production of embryos intended for internationalmovement.

4. Team personnel should be adequately trained in the techniques and principles of disease control. Highstandards 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 inspectionby 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 andthe production and storage of embryos.

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13 Stringfellow D.A. & Givens M.D. 2000.Preventing disease transmission through the transfer of in vivoderived 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 thEdition. (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.

16Thibier 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 vitroou 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. 4thEdition. (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. BritishVeterinary 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.

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