´ 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
6.4.1. Regulation measures: mastering the health risks associated with transmissible infectious
The fist significant 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 confirmed 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. confirm the disease free status of the young male.
The recent knowledge seems to confirm 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 271 –285
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 signifi- 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 fluids. 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 efficient
treatment equivalent to the one using trypsin for in vivo produced embryos on in vitro derived embryos
8. Recommendations associated with sanitary
