Livestock Production Science 62 2000 271–285 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 d ’Elevage et d’Insemination Artificielle, 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 first 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.: 1 33-14-353-5100; fax: 1 33-14-
and exchange of genetic material are accompanied
353-5101. ´
E-mail address : guerinaolclub-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. P I I : S 0 3 0 1 - 6 2 2 6 9 9 0 0 1 6 2 - 1
´ 272
B . Guerin et al. Livestock Production Science 62 2000 271 –285
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 scientific 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 qualified 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 define 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 271 –285 273
Table 2 In vitro production of embryos from non-fertile cows Guyader-Joly, unpublished
Oocytes collected after Holstein
Villard de Lans OPU
n 5 10, 24 sessions n 5 2, 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 5 2–3 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 first 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 5 26 donors,
Number Montbeliarde
Holstein 55 sessions
n 5 5, 12 sessions n 5 6, 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 271
– 285
Table 4 OPU-IVF embryos: Production compared in different systems
Animal Frequency
Superovulatory treatment In vitro maturation
Fertilization Culture
No. oocytes session No. transferable
of collect. embryos session
a
Fry, 1999 Prepubertal
1 3 month Sponge P4 5 4 inj. FSH
M199 1 FCS 1 hormones TALP 1 heparin
SOF 36.5
8.1 females
200 mg Folltropin Donnay et al., 1997
Heifers 2 3 week
No treatment M199 1 FCS 1 EGF
TALP 1 heparin SOF
3.9 1.7
Guyader-Joly et al., 1997 Pregnant
2 3 week No treatment
M199 1 FCS 1 hormones TALP 1 heparin
co-culture Vero 7
1.1 heifers
1 3 week 400 mg FSH stimufol
M199 1 FCS 1 hormones TALP 1 heparin
cells in B2 1 FCS 13
2.2 8 inj. decreasing
Bousquet et al., 1999 Cycled cows
Once every 400 mg FSH Folltropin
M199 1 FCS 1 hormones TALP 1 heparin
co-culture 9.5
4.7 2 weeks
8 inj. decreasing oviductal cells
in B2 1 FCS Looney et al., 1994
Non fertile Once every
No treatment M199 1 FCS 1 hormones
TALP 1 heparin co-culture BRL
6.2 0.96
cows 4–11 days
cells in M199 1 FCS Once every
30 mg FSH Schering M199 1 FCS 1 hormones
TALP 1 heparin co-culture BRL
8.6 1.38
4–11 days 6 inj.
cells in M199 1 FCS
a
Viability reduced after transfer in recipients.
´ B
. 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
