Livestock Production Science 62 (2000) 271285

www.elsevier.com/ locate/ livprodsci

A secure health status associated with the production and trade
of in vitro derived cattle embryos
a , a b
*
B. Guerin , Brigitte Le Guienne , M. Thibier
a
Union Nationale des Cooperatives dElevage et dInsemination Articielle, BP 65, 94703 Maisons-Alfort, France
b
AFSSA, BP 19, 94701 Maisons-Alfort, France
Abstract
The number of in vitro produced embryos used worldwide is increasing. In some countries such as Canada and the
Netherlands, genetics improvement programs use in vitro produced embryos extensively. Controlling the sanitary risks
associated with the production of these embryos relies on a different set of guidelines compared with the use of in vivo
produced embryos. There are particular risk factors: (1) the health status of the semen used for in vitro fertilization, (2) the
health status of the oocytes of the donor cow; (3) the freedom from contamination of the media and reagents used, and (4)
the environmental conditions associated with oocyte maturation, in vitro fertilization, cultivation and embryo transfer. Some
of these risks have already been studied, especially the principal pathogens found in bull semen (Brucella sp., Haemophilus
somnus, Campylobacter fetus, Leptospira sp. etc.). The oocytes may also be contaminated, either intracellularly (Campylo-
bacter fetus) or more frequently when virus (BHV-1, BVDV) or bacteria (Leptospira hardjo), Campylobacter fetus) located
in the ovarian follicle, are adsorbed onto the zona pellucida. Granulosa or cumulus cells, or even oviductal cells, can be a
source of infection when contaminated with virus (BHV-1, BVDV). For the production of in vitro embryos that present no
health risk, one must rely on using closely controlled and monitored cell lines, media and reagents that are guaranteed free of
products of animal origin. 2000 Elsevier Science B.V. All rights reserved.
Keywords: In vitro embryos; Sanitary risks; Oocyte maturation
1. Introduction then, these embryos have been used widely in
Canada, the United States and also in Europe, mainly
The rst in vitro embryos were produced in the in the Netherlands, Ireland, Italy and France (Table
early 50s (Dauzier et al., 1954), but the technique 1), some of the most advanced countries as far as
was really developed during the mid-80s, especially genetics are concerned.
in bovines, once the in vitro culture phase had been The constant improvements of these production
mastered. (Le Guienne and Thibier, 1988). Since protocols, which both enhance productivity and cut
costs together with improvements in embryo freezing
techniques, has led to the development of much
international embryo exchange. International trade
*Corresponding author. Tel.: 133-14-353-5100; fax: 133-14-
and exchange of genetic material are accompanied
353-5101.
E-mail address: guerinaol@club-internet.fr (B. Guerin) by the risk of disease transmission, particularly when
0301-6226/ 00/ $ see front matter 2000 Elsevier Science B.V. All rights reserved.
PI I : S0301- 6226( 99) 00162- 1
272 B. Guerin et al. / Livestock Production Science 62 (2000) 271285
Table 1
In vitro derived embryo production in Europe (Heyman, AETE 1998)
Countries IVF EMBRYOS
OPU embryos Slaught. embryos ET (fresh) ET (frozen) Frozen
a a
Belgium 355 21 ND ND 5907
France 405 628 190 36 6287
Ireland 1526 1315 209 2967
Italy 1793 558 391 2262 6381
Netherlands 3632 1778 951 10 000
UK 1009 227 280 138 8016
a
ND no data.
animals themselves are moved from one country to vitro produced embryos involves examining the
another. Some countries originally free of certain different steps of their production. Generally, those
diseases have unwittingly imported such pathogens risks refer to:
with livestock. As far as embryos are concerned, the
international scientic community, and the regula-
1. the oocytes, associated cells (cumulus and granul-
tory authorities of a large number of countries have
osa cells) and follicular environmental conditions
looked closely at these health risks associated with
2. the semen used for oocyte fertilization
the international exchange of genetic material.
3. the sanitary quality of the environment and re-
Much research was originally undertaken on in
agents used to cultivate those embryos and man-
vivo derived embryos, and this has led to the
ipulate them
publication of precise protocols, published in the
4. storage and shipment conditions
latest issue of the International Embryo Transfer
Society Manual (Stringfellow and Seidel, 1998).
The current document has three different objec-
Sanitary safety associated with embryo transfer was
tives:
so obvious that this technique was qualied as the
safest way to exchange genetic between countries or
1. to present the protocols used worldwide to gener-
continents (Thibier, 1990a).
ate in vitro produced embryos
As far as in vitro-produced embryos are
2. to identify the different parameters, which might
concerned, it soon became evident that such embryos
cause health risks
could require a different set of rules from in vivo-
3. to propose rules that can guarantee the sanitary
derived embryos. The biophysical characteristics of
quality of the embryos produced in these con-
the zona pellucida as well as the different production
ditions.
steps of these embryos are indeed peculiar, and
explain why the risks associated with their handling
are different.
From a strictly sanitary point of view, in vitro- 2. Production of in vitro-derived embryos
derived embryos could however be more advantage-
ous than in vivo-produced embryos because their The in vitro produced embryos can be obtained
sanitary quality can in theory be better controlled. It from oocytes collected from donor females, after
is indeed possible to control all parameters at stake ovum pick-up (OPU) or from slaughterhouse
in the making of such embryos with a certain degree ovaries. The follicles of 5 to 8 mm of diameter are
of reliability and to dene the guidelines to be manipulated with the help of a sterile needle. After
followed at each step in order to avoid any risks. collection the oocytes are immediately put in a
So, the analysis of health risks associated with in maturation medium.
B. Guerin et al. / Livestock Production Science 62 (2000) 271285 273
Table 2
In vitro production of embryos from non-fertile cows (Guyader-Joly, unpublished)
Oocytes collected after Holstein Villard de Lans
OPU (n 510, 24 sessions) (n 52, 5 sessions)
Number Average/ Number Average/
session session
Collected oocytes 315 13.1 157 31.4
Inseminated oocytes 269 11.2 114 22.8
Developed embryos 118 4.9 43 8.6
Transferred embryos 72 3.0 21 4.2
% of developed embryos 43.9% 37.7%
% gestation after 35 days 65.3% 66.7%
% gestation after 90 days 61.1% 57.1%
2.1. Production from oocytes donors collected 2.1.1. Superovulation treatment and pick-up period
The pick-up of oocytes can be performed several after ovum pick up (OPU) session
times a week (n 523) on females, without any
ovarian stimulation. Periods between pick-up ses- The oocytes can be harvested by OPU from pre-
sions can be more important when females are
pubertal females (Yang et al., 1997; Fry, 1999)
superovulated. The treatment consists of injecting
heifers or cows (Donnay et al., 1997; Lacaze et al.,
eight decreasing doses of FSH, whose concentration
1997). An interesting development of this technique
depends on the age of the animal: generally 70% of a
led to picking oocytes up from pregnant cows during
complete dose for cows, 50% for heifers and 25% or
the rst third of the gestation period (Guyader-Joly
less for pre-pubertal females.
et al., 1997).
The stimulation treatment seems to be mandatory
One of the most outstanding applications is its
for pre-pubertal animals in order to increase the
utilization to produce embryos from non-fertile
number of oocytes picked up: 26.4 oocytes for
females (Looney et al., 1994; Hasler et al., 1995;
sessions after FSH treatment vs. 3 oocytes picked up
Guyader-Joly, unpublished, Table 2).
Table 3
In vitro derived embryo production on pregnant and non-pregnant cows (from Guyader-Joly et al., 1997)
a b
Oocytes collected Non pregnant cows Pregnant cows
by OPU Result / session Result / session
Number n 526 donors, Number Montbeliarde Holstein
55 sessions (n 55, 12 sessions) (n 56, 10 sessions)
Collected oocytes 871 15.161.7 308 13.163.8 15.462.5
Inseminated 680 12.460.9 224 7.862.3 11.962.2
oocytes
Developed embryos 268 4.960.5 71 3.361.2 3.161.4
Transferred 166 3.160.4 46 2.360.9 1.961.0
embryos
% of developed 39.4% 38.1% 26.1%
embryos
% of gestation after 56% 37% 52.6%
35 days
a
Collection once a week, with superovulatory treatment.
b
Collection every two weeks.
274 B. Guerin et al. / Livestock Production Science 62 (2000) 271285
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B. Guerin et al. / Livestock Production Science 62 (2000) 271285 275
without any treatment (Mermillod et al., 1997). On Day 7, the embryos are checked on mor-
The use of FSH enables to stretch the period phological criteria and ranked in three categories:
between sessions without reducing the number of morula, blastocystes or degenerated embryos.
embryos produced during a given period. According
to Lacaze et al. (1997), the number of oocytes picked 2.1.3. Factors of variation
up and producing embryos are respectively 13.8 and The oocytes donor is one of the main factors of
1.8 for females picked up twice a week without variation, since the number of embryos produced for
ovarian stimulation versus 15.1 and 2.1 for the same each session may vary depending on the female from
animals picked up once a week after superovulation. 0.7 to 10.9 (P ,0.0002). As far as males used for
The same type of treatment applied to pregnant fertilization are concerned, the variation is less
heifers enabled them to produce also the same important and varies from 1.3 to 9.2 embryos
number of embryos per week (Guyader-Joly et al., produced per session (P ,0.001) (Twagiramungu et
1997) without disturbing the process of gestation al., 1999).
(Table 3). According to Hagemann et al. (1999), the size of
The period in between two collections may also be the picked up follicles can also be another factor,
two, three or four weeks (Bousquet et al., 1999). The given that the number of embryos produced from
use of ovarian stimulation on nonfertile females also oocytes obtained from follicles from 3 to 5 mm in
enables one to increase the number of oocytes picked diameter is signicantly higher than the one from
up (8.6 compared to 6.2) as well as the number of follicles from more than 6 mm.
embryos produced per session (1.38 vs. 0.96) Another inuential factor may also be the system
(Looney et al., 1994). of cultivation. Indeed, the number of embryos pro-
One of the benets of using the ovarian stimulat- duced for each session varies from 1.05 after co-
ing treatments is the reduction of the number of culture with BRL cells to 1.28 after culture in
sessions needed to produce a dened number of the SOF environment (P ,0.001, more than 1000
embryos, helping thus to cut production costs by sessions analyzed, Merton and Mullaart, 1999).
42% (whenever females are used once a week after
stimulation, instead of twice a week without treat-
ment). 3. Production from slaughterhouse ovaries
2.1.2. Protocols of production of in vitro embryos The gamete cells can also be picked up in the
The conditions of both in vitro maturation and slaughterhouse after evisceration, which enables one
fertilization are almost similar between the different to access the ovaries easily. In this case, ovaries are
teams (Table 4). The in vitro maturation environ- transported to the lab in phosphate buffer saline
ment widely used is the M199 mixed with fetal calf (PBS) kept under control at 258C. The cumulus
serum, hormones and growth factors if necessary. oocytes complex is taken out from 2 to 6 mm
Most of the production teams use the Tyrodes diameter follicles, and treated as for oocytes obtained
medium containing albumin, lactate and pyruvate after OPU sessions.
(TALP) to perform the in vitro fertilization with the This operation can lead to the recovery of several
presence of heparin (Ball et al., 1983; Parrish et al., tens of gamete cells for each ovary.
1986). However, the techniques of in vitro culture
vary from co-cultivation with the cells of the oviduct
or with cell lines (Vero cells or Buffalo Rat Liver 4. Sanitary risks associated with donor females
BRL) to cultivation in a semi-dened environment
such as the synthetic oviduct uid (SOF) or possibly 4.1. Sanitary risks linked to the follicular and
with the Rosenkrans medium CR1 or the potassium peri-oocyte environment
synthetic oviduct medium (KSOM). These are per-
formed under mineral oil (Takahashi and First, The risk is mainly linked to the oocytes them-
1992). selves, along with their follicular environment, which
276 B. Guerin et al. / Livestock Production Science 62 (2000) 271285
includes the cells of the cumulus and those of be taken so as to be able to avoid contamination
granulosa as well as the follicular liquid. Parallel to from one herd to another or within the same herd
this, the hygiene of collection is also a risk factor, from one animal to another.
which one has to master.
The pick-up of oocytes is always associated with
the presence of cells and biological uids, which 5. Hygienic rules and sanitary precautions
potentially present a risk for contamination. At the
time of collection, follicular uid and cells from 5.1. Hygienic rules
ovarian tissue are always mixed with cumulus and
granulosa cells. Rules to be followed for the collection of oocytes
The contamination of all these cells is possible and by OPU have been described elsewhere (Nibart et
has been demonstrated especially in the case of al., 1998). They are linked to material and equipment
infection by bovine viral diarrhea virus, BVDV, that must be perfectly adapted and cleaned and
(Avery et al., 1993; Bielanski and Dubuc, 1994; permanently ready to use.
Booth et al., 1992,1995) and by the infectious bovine Briey these rules address the following:
rhinotracheitis (IBR) virus (Guerin et al., 1989,1997;
Bielanski et al., 1993). Intra-vaginal probe, support and needle guide
must be sterile
4.2. Sanitary risks associated with the oocytes Needle must be disinfected and rinsed with a
sterile physiological solution before use
The major risk is associated with the intrafollicular No equipment should be ever used for more than
contamination of the oocyte that theoretically can be one donor without disinfecting
either cellular or supercial by absorbing pathogenic Collection liquid (PBS) unit must be conned to
agents onto the zona pellucida. The oocyte intracel- one individual
lular contamination has only been suggested for C. Technicians must be dressed with clean clothes
fetus (Bielanski, 1994) within the frame of ex- and boots
perimental contamination, which remains rather dif- Protection gloves must be disposable
ferent from the conditions of natural infection. Filter membranes used for oocytes must be sterile
BVDV and bovine herpes virus 1 (BHV-1) are and used only for one donor
undoubtedly the pathogenic agents most widely Oocytes must be kept in sterile ask containing
found in the oocytes environment (Bielanski et al., sterile medium
1993; Guerin et al., 1997). These viruses easily Transportation of the oocytes to the lab must be
adhere to the zona pellucida of the oocytes or the done in adequate conditions necessary to avoid
embryos (Singh et al., 1982; Guerin et al., 1989; any contamination.
Bielanski and Dubuc, 1993).
5.2. Sanitary precautions
4.3. Sanitary risk associated with ovum pick-up
The health status of the herd of origin of the donor
OPU is a very important step that must be handled cow is important. It should be free of contagious
using sophisticated hygienic procedures. A contami- diseases and should comply with the sanitary stan-
nation at this step can have a very detrimental effect dards of the country. At least, the herd must be free
and lead to an important reduction in terms of of tuberculosis and brucellosis. The donor female
embryo production. should also be free of clinical signs of contagious
The practical organization for in vitro derived diseases (Nibart et al., 1998).
embryos production means that frequently several As an example specic requirements can obvious-
donor females are collected at the same day and ly be set out so that no problems occur with IBR-IPV
perhaps originating from different herds. or BVD between different herds. In brief, the health
Under these conditions, hygienic precautions must surveillance of the donor animals relies on two blood
B. Guerin et al. / Livestock Production Science 62 (2000) 271285 277
Table 5
samplings, 23 weeks apart, the second one being
Percentage of hatched embryos after cultivation of frozen embryos
done at the time of the oocyte collection. In addition
in ethylene glycol in two different media (Guyader-Joly, un-
an aliquot of the follicular uid can be tested for
published)
relevant pathogens and particularly for IBR and
BSA A substitute
BVD viruses.
Number of cultivated embryos 37 49
Number of hatched embryos 22 31
Percentage 59.5% 63.3%
6. Sanitary risks associated with the production
of in vitro-derived embryos
oocytespermatozoaco-culture cells system. So, for
These risks are linked to the different steps example, complex culture media are now exclusively
associated with the production of in vitro embryos: made from amino acids from a plant origin, and it
maturation, fertilization, culture, storage and also has no detrimental effect on either the results of
with the conditions applied to handle them, which fertilization or the in vitro development rates
concerns material and culture media. (Guyader-Joly, unpublished Table 5).
Later on, the operations of transfer will also have The value of adding antibiotics to those media lies
to be performed in the best conditions as far as in the fact that it removes the pathogens (S. agalac-
hygiene and asepsis are concerned, by following the tiae, A. pyogenes, E. coli ) that could accidentally be
recommended classical guidelines very strictly associated with the oocytes or embryos during ovum
(Mapletoft and Stookey, 1998). pickup or culture phases (Otoi et al., 1992a,b).
One has to take great care in preparation of those
6.1. Environmental factors additives to the media because it has been demon-
strated that these products could easily be contami-
Environmental factors will be of capital impor- nated especially by BVD, parainuenza, blue tongue
tance during all the steps in the making of in vitro- and herpes viruses (Rossi et al., 1980; Van Soom et
produced embryos. One will have to make sure that al., 1994; Guerin et al., 1997; Brock, 1998). How-
labs, working area, incubators, and all devices in ever, the contamination of serum batches by BVD
contact with embryos are perfectly clean and handled virus can be associated with development rates that
with hygienic care. do not signicantly differ from those obtained from
In specialized labs, individuals units are set aside virus free serum (Guerin et al., 1997). This fact
for particular tasks with restricted access. The most stresses the basic necessity of the best quality serum.
advanced set-up also contains a laminar ow The risk of contamination from non-conventional
chamber with close attention to cleaning and dis- agents such as prion has also been thought of. The
infecting procedures. conclusions of very complete analysis seems to
indicate however that it is rather low (Wrathall,
6.2. Culture media 1999).
Some current research is looking at the technique
They must be able to provide the physiological of substituting macromolecules (sodium hyaluronate,
needs to the embryo so they will vary in their polyvinyl alcohol, polyvinylpyrrolidone, VF 5) hav-
composition depending on whether they are used for ing surfactant activity to reduce the health risk
maturation, fertilization, culture, or preservation of (Guyader-Joly et al., 1992; Palasz et al., 1993; Seidel
the embryos. Usually, they contain products from et al., 1990; Palasz et al., 1995).
animal origin, especially bovine oestrus serum, fetal
calf serum, bovine serum albumin or growth factors, 6.3. Contamination of co-culture cells
such as numerous amino acids of animal origin.
Nowadays, one tends to avoid using products from Oocytes are often cultivated with cumulus cells
animal origin as far as one can, at least within the during maturation. Later on zygotes may be co-
bound compatible to maintain the viability of the cultivated with oviductal cells, which can either
278 B. Guerin et al. / Livestock Production Science 62 (2000) 271285
belong to the oocytes donor (homologous system) or tion, culture) and subsequently lead to the contami-
come from cows from slaughter (heterologous sys- nation of the embryo nally produced (Guerin et al.,
tem). 1992; Bielanski et al., 1992a,b,c). However, a rel-
The contamination of these cellular systems was evant treatment with hyaluronic acid during the
shown for bacteria (Bielanski and Stewart, 1996) and preparation phase can reduce or denitely eradicate
also for the BVD and BHV-1 viruses, which are this contamination (Bielanski et al., 1992a,b,c).
frequently found in these conditions in Canada, the The use of bull sperm at this stage has to be
United States or France (Booth et al., 1992,1995; associated with very strict sanitary quality as far as
Bielanski et al., 1993; Guerin et al., 1997). The rate well-identied pathogens are concerned.
of isolation appears to relate to the importance of the However, the semen also needs to be prepared
disease in the individual countries. under strict aseptic conditions despite the fact that
The use of cell lines (BRL or Vero), where the bull sperm is never sterile from a bacteriological
controlling the absence of any viral, bacterial (among point of view. This is mainly due to:
which are the mycoplasmas) or fungi contamination
is easy to perform, enables one to reach the required the multiple microorganisms found in the ejacu-
quality level, and to operate in totally secured lates
disease free conditions. One might also consider or an abnormal location in the genital tract
getting rid of the co-culture phase by using SOF (accessory glands, testicles)
(totally synthetic medium) during this step. These or their presence as normal contaminant in the
SOF could be controlled as required and would urethra, preputial cavity or penis
ensure totally secure higher sanitary conditions. or because they may be present in the environ-
ment during the pick-up (collection room).
6.4. Sanitary risks associated with semen
The use of egg yolk (fresh egg or preparation
The production of in vitro embryos involves a made from industrial egg yolk) frequently leads to
fertilization step of the oocytes in extremely well the presence of bacterial species of avian origin in
controlled conditions. Some experimental work has the sperm. The latter are not killed by the antibiotics
shown (Barlow et al., 1986; Guerin et al., 1992; usually added to the extenders (gentamicin, penicil-
Bielanski and Loewen, 1994) that bulls persistently lin, streptomycin, linco-spectinomycin).
infected by BVDV shed virus in semen. Similar The sperm microora is made up of three kinds of
observations have been published for the BHV-1 and microorganisms: (1) the permanent pathogenic
for many other pathogenic agents. Some viruses agents: in this group are found the pathogenic
might even penetrate with the spermatozoa and species such as Brucella sp., Mycobacterium tuber-
therefore induce contamination of the embryo during culosis and paratuberculosis, Campylobacter fetus
the fertilization phase (Nussbaum et al., 1993). etc.) and some viruses (FMDV, BVDV, BHV-1,
Some bacterial species such as Stenotrophomonas EBLV, BTV etc.) (Afshar and Eaglesome, 1990); (2)
maltophilia, Pseudomonas putida, P. aeruginosa, A. the opportunistic pathogenic agents: in this group are
calcoaceticus or Flavobacterium have been iden- classied the bacteria species called the opportunistic
tied in some infertility cases based on in vitro pathogenic agents such as Escherichia coli, Staphy-
embryos production (Stringfellow et al., 1997a,b; lococcus aureus, Streptococcus faecalis, Pseudo-
Lee et al., 1997). monas aeruginosa, Haemophilus somnus etc. (Bar-
As detailed above, the spermatozoa used for the in tlett et al., 1976; Wierzsbowski, 1981; Humphrey et
vitro fertilization phase go through a special treat- al., 1982; Parez and Guerin, 1983), (3) the sap-
ment (swim up) in contact with antibiotics, which do rophytic microorganisms: in this group are classied
not necessarily eliminate all the pathogenic agents all bacteria species that are commensal in the natural
like bacteria or viruses. cavities of cattle as well as the intestinal species
In these conditions, a contamination with BVDV found in fecal material and therefore also in the
can occur during one of the in vitro phases (matura- bedding, which is the main source of the contamina-
B. Guerin et al. / Livestock Production Science 62 (2000) 271285 279
tion of the preputial cavity: Micrococcus, Staphylo- used to produce semen, bulls will be regularly and
coccus, Corynebacterium sp., Enterobacteriaceae, individually tested at least every year for several
Bacterodes sp., Clostridium etc., (Bonadonna, diseases such as tuberculosis, brucellosis, leucosis,
1971). IBR-IPV, Campylobacteriosis, Trichomoniasis.
From a bacteriological point of view, it is almost
impossible to have a totally sterile semen even if 6.4.2. Hygienic measures and lab best practices:
some antibiotics are used in the extenders. These mastering the microbiological quality of semen
antibiotics that some protocols require to add to the In order to produce high bacteriological quality
fresh semen before dilution rather than adding it to semen, some measures are of fundamental impor-
the ready made extenders, never have a 100% tance:
bactericidal activity on the bacterial ora of the
semen whatever bacterial species are considered. As the hygiene of the bull and the bull pen oor
a matter of fact, semen sterilization by addition of the hygiene of the collection (cow, collection
substances with antibacterial activity is an illusion. room, collector)
With regard to viruses, the antibiotics have no the treatment of the semen (sterile glassware, high
impact on them and it is therefore impossible to bacteriological quality extenders)
think about eradicating them or inactivating them the clean state and hygiene of the lab and the
through this process. devices used to handle the semen
Because of this, mastering the health risk associ- the staff, which has to be competent and trained
ated with the semen has to be dealt with by good in hygiene and disinfecting techniques
management. Most of this is incorporated into the working spaces (cleaned from a bacteriological
International Sanitary Guidelines and especially in point of view)
the European document (EEC Directive, 1988; the containers, which have to be cleaned and
Thibier, 1990b). regularly disinfected.
In order to improve the quality of the semen, some
centers have committed themselves to implementing The bacteriological quality of the liquid nitrogen
a quality audit. One of its main objectives is to used is also one of the important parameters. To this
reduce the contamination of the ecosystem associated extent, recycling nitrogen from one container to
with the preparation of semen. Some measures, another must not be done.
which are among the labs best practices, are listed
and are the foundation of the specication document
for semen production. 7. Interactions between pathogenic agents and
in vitro derived embryos
6.4.1. Regulation measures: mastering the health
risks associated with transmissible infectious The st signicant successes regarding in vitro
diseases derived embryo production date back from the late
These measures are in accordance with the fun- 80s (Le Guienne and Thibier, 1988). This time gap
damental principal of the careful selection of the compared with in vivo produced embryos explains
source of young sires for AI. The young male has to why there are so few researches about the interac-
be selected either from a herd free of disease or from tions between pathogenic agents and in vitro derived
a dam free of disease. It also has to be conrmed as embryos nowadays.
free of disease in its original herd before being The main principles previously set for in vivo-
accepted as a donor sire. A batch of health tests will produced embryos may also be applied to in vitro
be done before entering the collection center, over a derived embryos. One should not directly extrapolate
period of isolation that may vary from 30 to 60 days from an agent to another or also from a species to
depending on the regulations. This will help to another as rightly stated by Hare (1985).
conrm the disease free status of the young male. The recent knowledge seems to conrm that in
Later on in the process and as long as they are be vitro-produced embryos are much more vulnerable to
280 B. Guerin et al. / Livestock Production Science 62 (2000) 271285
Table 6
In vitro fertilization, cleavage and development rates of embryos after use of BVDV infected semen (from Guerin et al., 1992)
Groups Non-infected semen Infected semen
a b c
Fertilization (%) 61/ 64 (95.3%) 61/ 81 (79%)
b c
Cleavage (%) 56/ 61 (91.8%) 47/ 64 (73.4%)
b c
Blastocyste/ cleavage (%) 11/ 56 (19.6%) 1/ 47 (2.1%)
a
Estimated after observation of cleaved embryos or number of pronuclei after staining.
b
Author, please supply footnote.
c
Author, please supply footnote.
pathogenic agents than in vivo-derived embryos, After experimental infection of in vitro derived
mainly because of the differences in the structure of embryos by BHV-1 or BVDV (Guerin et al., 1990;
the zona pellucida that allows the adsorption of Bielanski et al., 1993) or by Leptospira hardjo
pathogenic agents. This peculiar characteristic is (Bielanski and Surujballi, 1996) embryos are con-
very important when assessing the risks associated taminated by these agents. Moreover a very signi-
with these embryos (Shi and Wrathall, 1989). Avail- cant reduction in the number of transferable embryos
able researches so far were done either on slaughter- occurred in these conditions with BVDV (Table 6)
house ovaries from all coming cows, some of them (Hare, 1986; Bielanski and Hare, 1988; Allietta et
being naturally infected with BVDV, or after an al., 1995; Bielanski and Dubuc, 1995; Stringfellow et
experimental infection of donor cows. al., 1997a,b).
After experimental or natural infection by BHV-1 Nevertheless, in special conditions, it is very
and a subsequent dexamethasone treatment, in vitro- interesting to note that embryos seem to have some
derived embryos and oviductal cells are contami- noticeable antiviral activity especially directed
nated (Guerin et al., 1989, 1990; Bielanski and against BVDV (Bielanski and Loewen, 1994;
Dubuc, 1994). Experimental contamination of the Zurovac et al., 1994; Bielanski and Dubuc, 1995;
oocytes during the maturation phase also leads to a Palma et al., 1996) and for BHV-1 (Van Roose et al.,
contamination of the embryos (Bielanski et al., 1997). Similar activity is also observed after ex-
1987). perimental infection of donor cows with BHV-1
These surveys have clearly shown that the sanitary (Guerin et al., 1997, unpublished).
control of the different phases of embryo production This particular effect and the low number of viral
could be wisely done on maturation and culture particles surrounding embryos could explain why the
uids. It has also proven that such a control has a BVD is not transmitted after embryo transfer of in
major interest as much for the virus as for bacteria vitro derived embryos collected from viremic donors
(Stringfellow et al., 1983; Brock et al., 1991; Bielan- (Van Soom et al., 1994).
ski et al., 1994) since there seems to be no efcient
treatment (equivalent to the one using trypsin for in
vivo produced embryos) on in vitro derived embryos 8. Recommendations associated with sanitary
(Avery et al., 1993; Tsuboi and Imada, 1996; Bielan- guarantees linked to the production of in vitro
ski et al., 1997). derived embryos
Sanitary washes recommended for in vivo em-
bryos seem to have more than a limited efciency for The principles stated would therefore be strictly
in vitro derived embryos (Bielanski et al., 1989; associated with the production of in vitro derived
Zurovac et al., 1994; Bielanski and Jordan, 1996; embryos and the guarantees that would lead to a
Trachte et al., 1997). worldwide protection of the breeders. No importer
Disinfecting procedures with photosensitive agents would like to introduce diseases when genetic needs
(haematoporphyrin, haematoporphyrin derivative or are required and imported. Consequently the sci-
thiopyronine) have been successfully used to inacti- entic veterinary community through the Internation-
vate BHV-1 and BVDV (Bielanski et al., 1992a,b,c). al Embryo Transfer Society published a manual of
B. Guerin et al. / Livestock Production Science 62 (2000) 271285 281
technical recommendations aimed to dene interna- laboratory as it has been done in France for 10
tional rules related to handling and production of in years in cattle (Thibier and Guerin, 1993).
vitro-produced embryos. Two chapters are especially
linked with transmission of diseases through in vitro-
8.2. Sanitary status of donor animals
produced embryos (Bielanski, 1998) and sanitary
rules (Nibart et al., 1998). In addition, one specic
Two distinct cases must be here considered,
appendix to the OIE International Animal Health
according to the origin of the oocytes: slaughter-
Code (4.2.2.5.) has been approved giving guidelines
house ovaries vs. OPU. In the rst case, the sanitary
to the Chief Veterinary Ofcers (OIE, 1992).
status of the herd of origin must be known at the
Application of these rules is targeted to facilitate
latest before transfer so that embryos could be
international trade of these embryos in association
destroyed if all necessary guarantees are not pro-
with a very high level of sanitary guarantees.
vided. Since such productions are often conducted in
several batches of embryos obviously not coming
from a single cow, a precise identication of the
8.1. The concept of ofcially approved embryo
group of donor cows for a given batch of embryos is
production teams
required so that tracability can be perfectly estab-
lished and used if required. If oocytes are obtained
One of the main principles is to authorize embryo
from well-known donor animals, it is easy to get
production only through a team of well-trained
guarantees on the sanitary status of the herd of origin
technicians and veterinarians that must be ofcially
so that animals can be submitted to complementary
approved by a veterinary authority (Thibier, 1993).
sanitary controls.
This system has been rst set up for bovine in
vivo-derived embryo transfer and is completely
8.3. Washing procedures
adaptable to in vitro embryo production. Given the
peculiar characteristics of in vitro-produced embryos,
During cultivation that must be conducted under
distinct authorizations would be required if a single
sterile conditions, saprophytic bacteria that can at-
team wishes to have both activities approved simul-
tach to the zona pellucida can accidentally contami-
taneously. The system has been accepted worldwide
nate media. The 10 sanitary washes done in PBS and
by IETS, OIE and by the European Union as well.
the 100-fold dilution in each bath will reduce these
Four conditions are needed for a team to be ofcially
bacterial problems (IETS Manual, 1998). Trypsin
approved by the veterinary authorities:
can be added although not mandatory because of the
lack of data showing its efciency in the in vitro
the team should be supervised by a well-trained
systems. Choice and composition of media especially
veterinarian upon sanitary and technical proce-
in term of products of animal origin should also be
dures to produce such embryos
particularly supervised to avoid unexpected contami-
the team should be able to work in satisfactory
nation.
conditions both in terms of housing and equip-
ment, especially those required for the laboratory
8.4. Sanitary control of media and co-culture cells
where oocytes are treated and embryos produced.
the team should commit itself to strictly follow
The sanitary controls that must be applied to
the procedures mentioned above (IETS Manual,
media and cells are fully described in the IETS
1998)
Manual. Briey:
the team should be regularly submitted to the
inspection of the veterinarian authorities and be
when used for in vitro embryo production, all also submitted to sanitary controls of the degener-
biological products must be strictly controlled and ated or non-fertilized embryos, maturation and
guaranteed free from microorganisms (virus, bac- ushed uids stored for this purpose. This materi-
teria or fungi). This particular point is especially al should be analyzed in an ofcially recognized
282 B. Guerin et al. / Livestock Production Science 62 (2000) 271285
useful for cell cultures used during the co-cultiva- 9. Conclusion
tion phase (Fray et al., 1995).
sera must be free from antibodies and be checked In vitro embryo production is a recent technique
before use (IETS Manual, 1998). They must be that offers a lot of opportunities in terms of increas-
heated at 568C during at least 30 min and be kept ing the value of breeding production or selection in
under freezing conditions (T # 2188C). cattle. The rate of embryos produced from slaughter-
permanent control of the inactivation procedures house oocytes or from OPU oocytes are now equiva-
used to inactivate pathogens throughout fabrica- lent. It allows this biotechnology to be more widely
tion: heating at 658C for at least 3 h, gamma used especially for pregnant cows with high genetic
irradiation at 2.5 mR, pH 5 treatment for 2 h, value, for infertile cows or for endangered breeds.
membrane ltration (0.22 mm). One of the major interests is also to use different
a control of the batches at the end of their bulls for fertilization of the oocytes collected from a
production (especially for their bacterial sterility) single cow.
addition of antibiotics is required even though an Success in in vitro embryo production depends on
entire bactericidal action is impossible to achieve the ability to perform series of technical steps, with
(gentamicin 2550 mg/ ml or kanamycin 50 mg/ unrelenting attention to detail, while minimizing
ml) (Bielanski and Surujballi, 1996; Bielanski factors reported to have a negative effect on the
and Stewart, 1996) outcome. Sound sanitary and hygienic measures at
all stages are vital to ensure success. The rules
Other associated measures lead to set up precise associated with in vitro-derived embryo production
rules in terms of importation for blood products of are connected with technical competencies, ofcial
cattle origin (EEC directive 96/ 405) or to authorize agreement and veterinary supervision of the team, a
importation only from free countries (New Zealand, perfect risk assessment depending on a lot of sci-
Canada) and under the condition that these products entic studies targeted to improve the sanitary
have been conveniently treated so that all guarantees guarantees associated with this technique.
related to the absence of pathogens of cattle origin A tight compliance with the rules published in the
can be provided. IETS Manual allows all users worldwide to be really
Only a strict compliance with the rules recom- efcient and to work with a high level of guarantee,
mended worldwide (IETS Manual, 1998; OIE, 1998) which has no equivalent in the world of breeding and
would allow users to have all sanitary guarantees animal reproduction because all steps are rigorously
associated with the production and use of in vitro controlled and performed by highly technical and
derived embryos. well-trained technicians working in well-equipped
laboratories and clean laboratory environment.
8.5. Ofcial recommendations The guarantees associated with in vitro-produced
embryos are higher than for in vivo-derived em-
In vitro embryo production procedures have been bryos. Such a comparison allows considering that the
ruled by the European Union in 1994. Rules and risk of transmission of infectious diseases through
technical procedures that must be complied with are this biotechnology is close to zero. The fundamental
clearly dened and rely on the rules published by concept of the safest way to exchange genetic from
OIE and IETS. European countries have to organize a sanitary point of view can be then extended to in
agreement of the ofcially authorized teams under vitro-produced embryos. In the future, an increase in
strictly controlled conditions above mentioned. the use of in vitro-produced embryos can be pre-
Hundreds of embryos are produced and traded dicted and will likely be associated with deep
throughout the world in strong sanitary security and freezing which is up to date, the limiting factor of
total reliability in terms of control of epidemiological the development of international trade of such em-
security. bryos.
B. Guerin et al. / Livestock Production Science 62 (2000) 271285 283
Bielanski, A., Dubuc, C., Hare, W.C.D., 1992b. Failure to remove
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