The present investigation was undertaken in order to
develop a method that would result in a synchronous in
vitro maturation of nucleus and cytoplasm of porcine
oocytes. A series of nine experiments was set up to
compare various types and combinations of sera and
porcine follicular fluid (pFF). A further important
investigative feature was the testing of the use of a
goat serum antibody against the maturation-inhibiting
hormone inhibin. In addition, two feeder layer systems
were tested as to their suitability for the in vitro
maturation of porcine oocytes and their in vitro
cultivation when fertilised. The oocytes were harvested
by cutting open ovarian follicles of slaughtered
peripubertal sows. They were washed three times in PBS
+ 10% foetal calf serum (FCS) and subsequently
transferred to a maturation medium (TCM199 + Protein
source/Feeder layer), where they were incubated at 39°C
and 5% CO2 for 46 hours. In Experiments 1 - 4, the
percentage of metaphase II oocytes that developed was
determined (maturation rate). In Experiments 1 and 2,
the cultured oocytes were stained with Giemsa and the
degree of expansion of the mucified cumulus oopherus
after maturation was assessed. The Giemsa stain was
replaced by Lacmoid stain in Experiments 3 and 4. In
Experiments 5 - 9, the oocytes matured in vitro were
fertilised in order to ascertain the efficiency of
their cytoplasmic maturation. In these latter five
experiments, both the rate of cellular division and the
integrity of the blastomeres' cytoplasm were
investigated (FDA staining). Also, the number of
zygotes containing two or m! ore embryonal nuclei were
counted (Hoechst A 33342 staining). In the first
experiment of this series, the influence of different
concentrations of FCS on the maturation rate of porcine
oocytes was tested (n = 192 - 258 oocytes/group). From
the results of this experiment, the recommended
concentration of FCS needed to be added to the basic
maturation medium (TCM199) was found to be 5% as this
resulted in the best maturation rate (48%), and an
optimally expanded cumulus (quality 1) in 47% of the
oocytes. A slightly higher degree of maturation (50%)
tended to be present when just 1% FCS was added, but
only 23% of the oocytes had an optimal expansion of
their cumulus oophorus. The generally used standard of
adding 10% FCS to the maturation medium provided only
middling success (39% maturation rate, 23% quality I
oocytes). The fundamental necessity of FCS in the
maturation medium was also confirmed by the results
from the first test group where no FCS was present:
just 27% metaphase II oocytes developed and only 17%
had an optimally expanded cumulus. Experiment 2 looked
at the effects of an inhibin-antibody-containing goat
serum on the maturation of porcine oocytes (n = 162 -
307 oocytes/group). The first control group grown with
only 10% pFF showed the best degree of maturation both
with respect to the rate of maturation and the
expansion of the cumulus oopherus (83% and 87%, resp.).
The groups of oocytes grown in maturation media
containing anti-inhibin antibodies did not grow so well
as those with pFF, but they tended to grow better than
those groups without this antibody (61% and 70% vs 64%
and 61%, resp.). As a second control group grown with a
heat-inactivated goat serum without the anti-inhibin
antibodies resulted in the poorest results (47%
maturation rate). It can be concluded that the addition
of the anti-inhibin antibody promotes maturationof
porcine oocytes The third experiment (n = 61 - 86
oocytes/group) looked anew at the use of various
concentrations of the anti-inhibin goat serum, whereby
in this series pFF was used as the basic protein source
and not, as in Experiment 2, the heat-inactivated goat
serum. All three media containing the anti-inhibin
antibody tended to have better maturation rates than
the three media without this antibody (71%, 71% and 65%
vs 58%, 67% and 68%, resp.). In Experiment 4, twelve
different test groups (n = 74 - 116 oocytes/group) were
set up in order to test not only the effect of
differing serum concentrations, but also the influence
of two feeder-layer combinations on the in vitro
maturation of porcine oocytes. All the test groups with
a feeder layer [Buffalo Rat Liver cells (BRL) or
porcine oviduct feeder layer (pEIL); Groups
2,3,5,6,8,9,11 and 12] showed significantly better
degrees of maturation than the test groups without
feeder layers (Groups 1,4,7,10). The best maturation
results were attained with 10% pFF and a BRL feeder
layer (88% metaphase II oocytes), where less than half
of these developed in the culture media without
serum/pFF and a feeder layer (40% metaphase II
oocytes). The test groups with both the
antibody-containing goat serum and pEIL or BRL tended
to grow better than those groups with the
non-heat-inactivated goat serum and a pEIL or BRL
feeder layer (81% and 78% vs 74% and 70%, resp.).
Maturation rates ! of 79% were achieved in the test
groups 11 and 12 grown with BRL or pEIL but without
serum or pFF, which shows that a feeder layer can
replace the function of serum in in vitro culture. In
order to test their degree of cytoplasmic maturation,
the oocytes in Experiment 5 were not stained directly
after maturation but were fertilised in vitro and their
rate of division was determined (n = 109 - 139
oocytes/group). Using the same test situation as in
Experiment 3, more oocytes were matured and fertilised.
Those test groups without the anti-inhibin antibody
which had shown a poor result in Experiment 3 showed in
this new test series a tendency for a higher rate of
division (32%, 32% and 34%). Those oocytes grown on
media with the lowest concentrations of anti-inhibin
exhibited an equivalent rate of division to this. In
comparison, the group which had exhibited the highest
maturation rate in Experiment 3 - the group grown on
the medium containing only 10% pFF - now showed the
lowest rate of division (25%). As the test situation
for these two experiments were the same, this result
indicates that there is a great discrepancy between the
degree of nuclear and cytopl! asmic maturation induced
by the various additives to the basic culture medium.
The degree of cytoplasmic maturation of the oocytes was
then assessed in Experiment 6 (n = 158-220
oocytes/group). The test situations in this experiment,
whereby the oocytes were cultured with a feeder layer
mirrored those set up in experiment 4. The best rates
of division occurred in the culture with 9% pFF, 1%
anti-inhibin and a BRL feeder layer (38%). The worst
results occurred in the culture without either a
protein source or a feeder layer. The rate of division
of all the other test groups lay between 25 and 32%. In
Experiment 7, a complex system of judgement for the
classification of the test groups was used (n = 84-137
oocytes/group). The rate of division, the integrity of
the cytoplasm and the number of nuclei after in vitro
fertilisation were assessed for every oocyte. The
design of this experiment was the same as for
Experiment 6. The worst results for all three criteria
were found in those test groups without a feeder layer.
A cultivation constellation of 9% pFF, 1% anti-inhibin
and a BRL feeder layer produced the best results for
the rate of division, vitality and the percentage of
zygotes with 2 or more nuclei (39%, 89% and 40%,
resp.). The cultures with 9% pFF, 1% anti-inhibin and
pEIL had a 35% rate of division, 94% vital oocytes and
50% fertilised oocytes containing 2 or more nuclei.
These two methods of cultivation can both, therefore,
be recommended for the in vitro maturation of porcine
oocytes. Experiment 8 considered the use of a feeder
layer not only for the maturation phase but also for
the subsequent culture of the embryo (n = 68-80
oocytes/group). The best results in both culture phases
were obtained with a BRL feeder layer system (66%
division rate, 84% vital oocytes and 78% oocytes with 2
or more nuclei). In comparison, the maturation and
embryo cultures without feeder layers exhibited a rate
of division of 18%, 48% vital oocytes, and 25% oocytes
with 2 or more nuclei. As in comparison to the
feeder-layer-supported embryo cultures, there were
significantly poorer results for all three parameters
in those cultures where a feeder layer was not used
during the embryonal culture phase, it may be concluded
that within the scope of this research work, feeder
layers have a positive effect on both these phases of
oocyte cultivation. In Experiment 9, in vitro matured
oocytes were compared with in vivo matured oocytes (n =
127-130 oocytes/group). The immature oocytes were
matured in vitro on oviduct feeder layers and then
cultivated, while the mature oocytes were collected
from superovulated sows. The success of the different
maturation systems were then tested by embryo transfer.
The mature oocytes in each group were fertilised in
vitro and the resulting embryos were implanted in
synchronised recipient sows. A viable pregnancy could
not be produced in any of the test groups.
| Identifer | oai:union.ndltd.org:uni-goettingen.de/oai:ediss.uni-goettingen.de:11858/00-1735-0000-0006-B6E6-E |
| Date | 20 May 1999 |
| Creators | Ropeter-Scharfenstein, Manuela |
| Source Sets | Georg-August-Universität Göttingen |
| Language | German |
| Detected Language | English |
| Type | doctoralThesis |
| Format | application/pdf |
| Rights | http://webdoc.sub.gwdg.de/diss/copyrdiss.htm |
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