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. Guerin et al. Livestock Production Science 62 2000 271 –285 275
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 oocyte’s 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 significantly 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 influential factor may also be the system Looney et al., 1994.
of cultivation. Indeed, the number of embryos pro- One of the benefits 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 defined 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 Tyrode’s 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-defined environment such as the synthetic oviduct fluid 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 271 –285
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 fluids, which 5. Hygienic rules and sanitary precautions
potentially present a risk for contamination. At the time of collection, follicular fluid 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 Briefly 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 superficial by absorbing pathogenic
• Collection liquid PBS unit must be confined 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 flask 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 specific 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
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. 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
