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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.

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