´ B
. Guerin et al. Livestock Production Science 62 2000 271 –285 277
Table 5
samplings, 2–3 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 fluid 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
oocyte–spermatozoa–co-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Guyader-Joly, unpublished Table 5.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, parainfluenza, 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 significantly 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 flow
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 271 –285
belong to the oocyte’s donor homologous system or tion, culture and subsequently lead to the contami-
´ come from cows from slaughter heterologous sys-
nation of the embryo finally 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 definitely 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-identified 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 microflora 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- classified the bacteria species called the opportunistic
tified 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 classified 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 271 –285 279
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
¨ Bacteroıdes
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 flora of the semen whatever bacterial species are considered. As
• the hygiene of the bull and the bull pen floor
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 lab’s best practices, are listed
and are the foundation of the specification document for semen production.
7. Interactions between pathogenic agents and in vitro derived embryos
