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In this study, we compared the developmental capacity of bovine haploid and diploid androgenetic and parthenogenetic embryos obtained by different methods. Androgenetic embryos were produced by piezo-intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF) of enucleated oocytes with or without subsequent pronuclear transfer from one haploid zygote to another. Parthenogenetic embryos were obtained by activation of matured oocytes by ionomycin combined with cycloheximide or 6-dimethylaminopurine (DMAP) treatment. Only few cleaved androgenetic haploid embryos were able to compact (2.7%) and to form blastocysts (1.8%), while significantly more haploid parthenogenotes underwent compaction (24-37%) and a minority developed to blastocysts at different rates, depending on the activation procedure (cycloheximide 3%, 6-DMAP 14.5%). By contrast, development to blastocyst of diploid androgenotes, cloned androgenetic embryos, and parthenogenotes (31%, 39%, and 43%, respectively) was similar to IVF control embryos (35%). Cell number on Day 7 was higher for IVF blastocysts and decreased in consecutive order in diploid androgenotes, diploid parthenogenotes, and haploid uniparental embryos. Following transfer of diploid androgenetic embryos, a pregnancy was established and maintained up to Day 28.

The aim of this article was to compare plasma estrone sulfate (E1SO4), clinical biochemistry, and milk yield of dairy cows carrying a female fetus from a bull (BULL) or from its clone (CLONE), evaluating also the relationship between the former variables and the birth weight of the newborn. Sixteen recipient dairy Friesian heifers (10 BULL and 7 CLONE) received a female embryo, obtained by in vitro embryo production and sexing by polymerase chain reaction with the semen of the BULL or the CLONE. Blood samples on all cows were obtained before feed distribution in the morning from jugular vein from 4 weeks before to 4 weeks after calving, to be analyzed for metabolic profile. The samples from late gestation were also analyzed for E1SO4 concentration. To separately assess the effect of calf birth weight (CBW), data were categorized as follows: low (46 kg; BWT-C). The plasma concentrations of β-hydroxybutyric acid (BHB, P=0.019), Na (P=0.002), Cl (P=0.026), strong cation-anion balance (P=0.020), total bilirubin (P=0.054), and α1-globulin (P=0.044) were higher in prepartum BULL recipients than those in CLONE, whereas BHB (P=0.021) and Mg (P=0.090) were higher in postpartum BULL recipients, while no differences were recorded in the remaining postpartum parameters. The CBW class had significant interaction with week of gestation on antepartum plasma estrone sulfate (P=0.021), whereas CBW per se affected antepartum plasma BHB (P=0.021), and nonesterified fatty acids (NEFA; P=0.011) being higher in BWT-C which also had the lower NEFA concentration during postpartum. Milk yield was unaffected by the sire used, both for quantitative and qualitative aspects. Cows carrying heavier fetus (BWT-C) had a different lactation affected by month compared with the other 2 CBW groups. From these results, there were no differences between BULL and CLONE recipients. Estrone sulfate, BHB, and NEFA may be used to predict CBW and provide different nutritional management during gestation.

Amniotic fluid (AF) is a source of multipotent mesenchymal stem cells (MSCs), very promising cells for tissue engineering in clinical application. The aim of this work was to isolate and characterize cells isolated from bovine AF as alternative sources of primitive multipotent stem cells in a species that could be a large-animal model for biomedical and biotechnology researches. Samples were recovered, at slaughterhouse, from 39 pregnant cows at different trimesters of pregnancy and cells were cultured in vitro. At passages (P) 3 and 7 differentiation towards chondrogenic, osteogenic and adipogenic lineages was induced. Flow cytometry analysis for CD90, CD105, CD73, CD44, CD34, CD45 and CD14 was performed, immunocytochemistry (ICC) for Oct4, SSEA4, α-SMA, Vimentin, N- and E- Cadherin and CK and qPCR analysis for OCT4, NANOG and SOX2 were carried out. The cell yield was significantly higher in the first trimester compared to the second and the third one (P < 0.05). Cells were isolated from 25/39 samples and cell population appeared heterogeneous. Two main cell types were identified in samples from all trimesters: round- (RS) and spindle-shaped (SS) cells. 17/25 samples showed both populations (mixed, MX). Both cell types showed MSC-markers and differentiation capability with some variability related to the passages. The SS-population also expressed low levels of stemness markers such as NANOG and SSEA4 but not OCT4. Bovine AF shows a heterogeneous cell population containing also MSCs, multipotent cells that represent an intermediate stage between embryonic stem cells and adult ones.

The aim of this study was to establish whether perturbed gene expression during cumulus oocyte development causes repeat breeding in cattle. In this study, a repeat breeder was defined as a normal estrous cycling animal that did not become pregnant after three inseminations despite the absence of clinically detectable reproductive disorders. Transcripts of genes extracted from cumulus oocyte complexes (COC) that were collected from three repeat breeder and three normally fertile Holstein Friesian heifers were compared. Up to 40 COC were collected from each heifer by means of repeated sessions of ovum pick up in the absence of hormonal stimulation; immediately plunged into liquid nitrogen; and stored at −80 °C until analysis. For each heifer, RNA was extracted from the pooled COC and hybridized on GeneChip® Bovine Gene Array (Affymetrix). Analysis of gene expression profiles of repeat breeder and control COC showed that 178 genes were differentially expressed (log2 fold change > 1.5). Of these genes, 43 (24%) were up-regulated and 135 (76%) were down-regulated in repeat breeder relative to control heifers. This altered pattern of expression occurred in genes involved in several cellular biological processes and cellular components such as metabolism, angiogenesis, substrate/ion transport, regulation/signaling, cell adhesion and cytoskeleton. From these, 13 genes potentially involved in cumulus oocyte growth were subjected to validation by qRT-PCR and nine genes (annexin A1, ANXA1; lactoferrin, LTF; interferon stimulated exonuclease 20 kDa, ISG20/HEM45; oxidized low density lipoprotein receptor 1, OLR1; fatty acid desaturase 2, FADS2; glutathione S-transferase A2 and A4, GSTA2 and GSTA4; glutathione peroxidase 1, GPX1; endothelin receptor type A, EDNRA) were confirmed to be differentially expressed. This study identified potential marker genes for fertility in dairy cattle.

The possibility to genotype embryos prior to implantation would have advantages for increasing the speed of selection of cattle. Reliable genotyping requires more DNA than can be obtained from biopsies of embryos, if they are to remain viable. Multiple displacement amplification (MDA) is a whole genome amplification technique used to increase the amount of DNA from biopsies for analysis. Reduced genome coverage resulting in Allele Drop Out (ADO) at heterozygous loci or missing genotypes are drawbacks of MDA.The present article describes the correlation between the input DNA quantity or embryo biopsy size and MDA success. Missing genotypes and ADO drastically increased when fewer than 30–40 cells or the genomic equivalents were used. However, embryo viability was found to be reduced if biopsied with more than 10 cells. Therefore, in vitro cell culture was investigated as a means to increase the number of cells available and the genotyping reliability.

Reproductive toxicity, with its many targets and mechanisms, is a complex area of toxicology; thus, the screening and identification of reproductive toxicants is a main scientific challenge for the safety assessment of chemicals, including the European Regulation on Chemicals (REACH). Regulatory agencies recommend the implementation of the 3Rs principle (refinement, reduction, replacement) as well as of intelligent testing strategies, through the development of in vitro methods and the use of mechanistic information in the hazard identification and characterization steps of the risk assessment process. The EU Integrated Project ReProTect (6th Framework Programme) implemented an array of in vitro tests to study different building blocks of the mammalian reproductive cycle: methodological developments and results on male and female germ cells, prostate and placenta are presented.

Infertility in cattle herds is a growing problem with multifactorial causes. Embryonic genotype and level of inbreeding are among the many factors that can play a role on reproductive efficiency. To investigate this issue, we produced purebred and crossbred bovine embryos by in vitro techniques from Holstein oocytes and Holstein or Brown Swiss semen and analyzed several cellular and molecular features. In the first experiment, purebred and crossbred embryos, obtained from abattoir oocytes, were analyzed for cleavage, development to morula/blastocyst stages, amino acid metabolism and gene expression of developmentally important genes. The results indicated significant differences in the percentage of compacted morulae, in the expression of three genes at the blastocyst stage (MNSOD, GP130 and FGF4) and in the utilization of serine, asparagine, methionine and tryptophan in day 6 embryos. In the second experiment, bovine oocytes were collected by ovum pick up from ten Holstein donors and fertilized with the semen of the respective Holstein sires or with Brown Swiss semen. The derived embryos were grown in vitro up to day 7, and were then transferred to synchronized recipients and recovered on day 12. We found that purebred/inbred embryos had lower blastocyst rate on days 7–8, were smaller on day 12 and had lower expression of the trophoblast gene PLAC8. Overall, these results indicate reduced and delayed development of purebred embryos compared with crossbred embryos. In conclusion, this study provides evidence that embryo genotype and high inbreeding can affect amino acid metabolism, gene expression, preimplantation development and therefore fertility in cattle.

The most successful development of interspecies somatic cell nuclear transfer (iSCNT) embryos has been achieved in closely related species. The analyses of embryonic gene activity in iSCNT embryos of different species combinations have revealed the existence of significant aberrations in expression of housekeeping genes and genes dependent on the major embryonic genome activation (EGA). However, there are many studies with successful blastocyst (BL) development of iSCNT embryos derived from donor cells and oocytes of animal species with distant taxonomical relations (inter-family/inter-class) that should indicate proper EGA at least in terms of RNA polymerase I activation, nucleoli formation, and activation of genes engaged in morula and BL formation. We investigated the ability of bovine, porcine, and rabbit oocytes to activate embryonic nucleoli formation in the nuclei of somatic cells of different mammalian species. In iSCNT embryos, nucleoli precursor bodies originate from the oocyte, while most proteins engaged in the formation of mature nucleoli should be transcribed from genesde novoin the donor nucleus at the time of EGA. Thus, the success of nucleoli formation depends on species compatibility of many components of this complex process. We demonstrate that the time and cell stage of nucleoli formation are under the control of recipient ooplasm. Oocytes of the studied species possess different abilities to support nucleoli formation. Formation of nucleoli, which is a complex but small part of the whole process of EGA, is essential but not absolutely sufficient for the development of iSCNT embryos to the morula and BL stages.

The best results of inter-species somatic cell nuclear transfer (iSCNT) in mammals were obtained using closely related species that can hybridise naturally. However, in the last years, many reports describing blastocyst development following iSCNT between species with distant taxonomical relations (inter-classes, inter-order and inter-family) have been published. This indicates that embryonic genome activation (EGA) in xeno-cytoplasm is possible, albeit very rarely. Using a bovine–pig (inter-family) iSCNT model, we studied the basic characteristics of EGA: expression and activity of RNA polymerase II (RNA Pol II), formation of nucleoli (as an indicator of RNA polymerase I (RNA Pol I) activity), expression of the key pluripotency geneNANOGand alteration of mitochondrial mass. In control embryos (obtained by IVF or iSCNT), EGA was characterised by RNA Pol II accumulation and massive production of poly-adenylated transcripts (detected with oligo dT probes) in blastomere nuclei, and formation of nucleoli as a result of RNA Pol I activity. Conversely, iSCNT embryos were characterised by the absence of accumulation and low activity of RNA Pol II and inability to form active mature nucleoli. Moreover, in iSCNT embryos,NANOGwas not expressed, and mitochondria mass was significantly lower than in intra-species embryos. Finally, the complete developmental block at the 16–25-cell stage for pig–bovine iSCNT embryos and at the four-cell stage for bovine–pig iSCNT embryos strongly suggests that EGA is not taking place in iSCNT embryos. Thus, our experiments clearly demonstrate poor nucleus–cytoplasm compatibility between these animal species.

The new European chemicals policy for the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) will most probably impose a dramatic increase in the number of animals required for reproductive toxicity testing. For this purpose, the development and validation of alternative methods is urgently needed in order to reduce the use of laboratory animals. The present study describes the inter-laboratory variability and the transferability assessment of an in vitro test able to identify chemical effects during the process of oocyte maturation in a bovine model. The test was developed/optimised within ReProTect, an integrated research project funded by the European Union, joining together 35 partners with complementary expertise in reproductive toxicology. Eight chemicals with well-known toxic properties were tested (benzo[a]pyrene, busulfan, cadmium chloride, cycloheximide, diethylstilbestrol, ketoconazole, methylacetoacetate, mifepristone/RU-486 and DMSO as solvent) on the in vitro maturation (IVM) assay in two well-trained laboratories using the established Standard Operating Procedures. The statistical analysis demonstrated the concordance of results across the laboratories and the reproducibility of the test. We therefore conclude that the IVM test could advance toward the process of validation as alternative in vitro method that, in combination with additional in vitro tests, can become part of an integrated testing strategy in order to predict chemical hazards on mammalian fertility.

The culture of early embryos in the surrogate xeno-oviduct was first developed in the early 1950s to allow transport of embryos at long distances. Later, it was applied to the study of culture requirements of the early embryo especially that of bovine origin. In this article, we review the data available on the culture of in vitro-matured and in vitro-fertilized embryos of Bos taurus, Sus scrofa, Equus caballus and Ovis aries in the surrogate sheep oviduct compared with data on in vitro culture in different media. Short-term and long-term cellular and molecular effects are described mainly for the bovine species where more extensive use of this technique has been made. A comparison with in vitro culture in various conditions and species indicate that embryos cultured in the sheep oviduct have close similarities to totally in vivo-derived embryos. The data provided demonstrate that the technique of in vivo culture in the surrogate sheep oviduct is versatile and allows a high rate of embryonic development in all species examined.

The dramatic increase in the number of animals required for reproductive toxicity testing imposes the validation of alternative methods to reduce the use of laboratory animals. As we previously demonstrated for in vitro maturation test of bovine oocytes, the present study describes the transferability assessment and the inter-laboratory variability of an in vitro test able to identify chemical effects during the process of bovine oocyte fertilization. Eight chemicals with well-known toxic properties (benzo[a]pyrene, busulfan, cadmium chloride, cycloheximide, diethylstilbestrol, ketoconazole, methylacetoacetate, mifepristone/RU-486) were tested in two well-trained laboratories. The statistical analysis demonstrated no differences in the EC50 values for each chemical in within (inter-runs) and in between-laboratory variability of the proposed test. We therefore conclude that the bovine in vitro fertilization test could advance toward the validation process as alternative in vitro method and become part of an integrated testing strategy in order to predict chemical hazards on mammalian fertility.

Many factors influence the efficiency of the in vitro embryo production technology in cattle but the most important are the physiological conditions of the donor and the culture protocols for oocyte maturation and fertilization and for embryo culture from zygote to blastocyst. Therefore, general factors such as age, body conditions and herd management play a pivotal role together with more specific factors such as reproductive soundness and ovarian cyclicity. Given that good quality and competent oocytes are available a complex series of processes, including oocyte maturation, fertilization and culture of the derived zygotes, must be completed to generate viable embryos.

Assisted reproductive techniques developed for cattle in the last 25 years, like ovum pick up (OPU), intracytoplasmic sperm injection (ICSI), and somatic cell nuclear transfer, have been transferred and adapted to buffalo and horses. The successful clinical applications of these techniques require both the clinical skills specific to each animal species and an experienced laboratory team to support the in vitro phase of the work. In cattle, OPU can be considered a consolidated technology that is rapidly outpacing conventional superovulation for embryo transfer. In buffalo, OPU represents the only possibility for embryo production to advance the implementation of embryo-based biotechnologies in that industry, although it is still mainly in the developmental phase. In the horse, OPU is now an established procedure for breeding from infertile and sporting mares throughout the year. It requires ICSI that in the horse, contrary to what happens in cattle and buffalo, is very efficient and the only option because conventional IVF does not work. Somatic cell nuclear transfer is destined to fill a very small niche for generating animals of extremely high commercial value. The efficiency is low, but because normal animals can be generated it is likely that advancing our knowledge in that field might improve the technology and reduce its cost.

Current European legislation for the registration and authorisation of chemicals (REACH) will require a dramatic increase in the use of animals for reproductive toxicity testing. Since one objective of REACH is to use vertebrates only as last resort, the development and validation of alternative methods is urgently needed. For this purpose ReProTect, an integrated research project funded by the European Union, joining together 32 partners with complementary expertise in the area of reproductive toxicology was created. The presented study will describe a battery of two tests that have been developed within ReProTect. The objectives of these tests are the detection of chemical effects on the bovine oocyte, during the processes of maturation and fertilisation. The protocols have been optimised and 15 selected chemicals were tested allowing the assessment of the intra-laboratory variability, the definition of a preliminary prediction model and the identification of the most predictive endpoint of the fertilisation test. After successful validation these tests should be part of an integrated testing strategy that combines the bovine model with additional in vitro tests that are able to detect effects on steroidogenesis, folliculogenesis and male fertility in order to predict chemical hazards on mammalian fertility., JRC.I.2-In-vitro Toxicology

This paper summarizes the major advances in farm animal assisted reproduction in the last 20 years with particular attention to the contributions of the authors. A main emphasis is on the biology of the oocyte, including a description of methods for isolation of developing follicles and culture of the corresponding oocytes. Milestones that have led to optimization of procedures for maturation of fully grown oocytes, fertilization, intracytoplasmic sperm injection and embryo culture in sheep, cattle, pigs and horses are described. The current status of nuclear transfer, cloning and embryonic stem cell generation and culture is also summarized for all major farm animal species. It is concluded that the manipulation of early development in farm animals is of crucial importance for agricultural purposes and that reproductive biotechnologies in farm animals are expected to play an increasing role in the next decades due to the growing demand for agricultural products from the emerging economies worldwide. In the biomedical field large animals represent increasingly important research models especially in the stem cell field for creating genetically modified animals for specific purposes. Finally, the successful translation of large animal research in the applied context requires solid science, long-term resource commitment from involved institutions, and vision, dedication and entrepreneurial skills from the scientists involved.

Histone modification genes in bovine embryos: The mRNA expression pattern of histone-related genes was determined in bovine oocytes and embryos. We compared immature and in vitro-matured oocytes, either before or after enucleation and activation, in vitro produced embryos (zygotes, 8-16 cell stages, blastocysts), embryos cloned with female or male donor cells; parthenogenetic embryos, and in vivo-derived blastocysts to detect deviations from the normal expression pattern. A sensitive semi-quantitative endpoint RT-PCR assay was used to reveal differences in histone deacetylation [histone deacetylase 2 (HDAC2)]; histone acetylation [histone acetyltransferase 1 (HATI)]; histone methylation [histone methyltransferases (SUV39H1, G9A)]; heterochromatin formation [heterochromatin protein 1 (HP1)]; and chromatin-mediated transcription regulation [zygote arrest 1 (ZAR1)]. With the exception of ZAR1, these mRNAs were present throughout preimplantation development. The relative abundance of mRNAs for histone methyltransferases (SUV39H1 and G9A) and for heterochromatin-associated protein (HP1) differed significantly before and after activation of the bovine embryonic genome. The similarity of HATI gene expression in 8-16 cell embryos and blastocysts suggests that histone acetylation is primarily affected by in vitro culture only prior to embryonic genome activation. HDAC2 gene mRNA expression was not affected by in vitro culture and/or cloning before and after activation of the embryonic genome. The donor cell line affected mRNA expression patterns of genes involved in reprogramming cloned embryos suggesting epigenetic dysregulation. Results show that both in vitro production and somatic cloning alter the mRNA expression of histone modifying genes in bovine embryos. Mol. Reprod. Dev. 75: 731743, 2008. (C) 2007 Wiley-Liss, Inc

Nuclear transfer (NT) is a complex procedure that requires considerable technical skills. Over the years attempts have been made to simplify the micromanipulations involved and to make the procedure more user-friendly. A significant step forwards has been the development of the zona-free NT methods. We have used zona-free NT with mechanical aspiration of the metaphase plate as a mean of enucleation, in a comparative approach with the conventional nuclear transfer zona-enclosed method in cattle, horse, sheep and pig. The absence of the zona considerably facilitates the enucleation step and significantly increases cell fusion success. On the other hand, the culture of zona-free NT embryos requires the embryos to be cultured individually or anyway separated from each other to avoid aggregation and also requires to prolong the in vitro culture up to the blastocyst stage before transfer. Blastocyst rate is equal or higher with zona-free method as compared to zona-enclosed method while survival after cryopreservation and development to term is comparable. In conclusion, our findings, together with published data, demonstrate that the zona-free system described in this paper can significantly increase the output of NT blastocysts over the conventional zona-enclosed system.

Embryonic stem cells differentiate into neuroectodermal cells under specific culture conditions. In primates, these cells are organized into rosettes expressing Pax6 and Sox1 and are responsive to inductive signals such as Sonic hedgehog (Shh) and retinoic acid. However, direct derivation of organized neuroectoderm in vitro from preimplantation mammalian embryos has never been reported. Here, we show that bovine inner cell masses from nuclear transfer and fertilized embryos, grown on feeders in serum-free medium, form polarized rosette structures expressing nestin, Pax6, Pax7, Sox1, and Otx2 and exhibiting interkinetic nuclear migration activity and cell junction distribution as in the developing neural tube. After in vitro expansion, neural rosettes give rise to p75-positive neural crest precursor cell lines capable of long-term proliferation and differentiation in autonomic and sensory peripheral neurons, glial cells, melanocytes, smooth muscle cells, and chondrocytes, recapitulating in vitro the unique plasticity of the neural crest lineage. Challenging the rosette dorsal fate by early exposure to Shh induces the expression of ventral markers Isl1, Nkx2.2, and Nkx6.1 and differentiation of mature astrocytes and neurons of central nervous system ventral identity, demonstrating appropriate response to inductive signals. All together, these findings indicate that neural rosettes directly derived from cloned and fertilized bovine embryos represent an in vitro model of early neural specification and differentiation events. Moreover, this study provides a source of highly proliferative neural crest precursor cell lines of wide differentiation potential for cell therapy and tissue engineering applications.

Mesenchymal stem cells (MSCs) reside in the bone marrow and have the potential for multilineage differentiation, into bone, cartilage, and fat, for example. In this study, bovine and porcine MSCs were isolated, cultured to determine their replication ability, and differentiated with osteogenic medium and 5-azacytine. Both bovine and porcine undifferentiated MSCs were electroporated and virally transduced to test the efficiency of genetic modification and the maintainance of differentiation ability thereafter. Nuclear transfer experiments were carried out with bovine and porcine MSCs, both at the undifferentiated state and following differentiation. Our results indicate that bovine and porcine MSCs have limited lifespans in vitro--approximately 50 population doublings. They can be efficiently differentiated and characterized along the osteogenic lineage by morphology, alkaline phosphatase, Von Kossa, oil red stainings, and RT-PCR. Electroporation and selection induce high levels of EGFP expression in porcine but not in bovine MSCs. Following genetic modification, MSCs retain their pluridifferentiation ability as parental cells. Cloned embryos derived from bovine and porcine undifferentiated MSCs and their derivatives along the osteogenic lineage give rise to consistently high preimplantation development comparable to adult fibroblasts.

This paper presents information on the evolution of sets of cloned heifers of Holstein breed in comparison to that of control heifers derived from artificial insemination (AI) in the same farm, as well as data on a set of cloned bulls and their semen characteristics. Preliminary observations on a group of calves sired by a cloned bull and offspring of cloned females are reported. Mean birth weight in the clone group (50 females) was statistically higher than that of 68 contemporary female controls obtained by AI (49.27 +/- 10.98 vs. 40.57 +/- 5.55 kg, respectively, p0.05). Growth rate was within normal values for Holstein heifers (from 0.7 to 0.8 kg/day) and daily gain was not influenced by the high or low birth weight of clones. Within animals of the same clone, variability of daily gain was reduced compared to their control counterparts. Semen production from three cloned bulls was within the parameters expected for young bull of the same age. A direct comparison of morphological analysis was made between the frozen thawed semen of the donor bull and of his three clones collected at the same age. The overall semen picture appeared within acceptable limits and the clones presented similar percentages of sperm abnormalities (80% of morphologically normal spermatozoa) as the donor. These preliminary results suggest no deleterious effect of cloning on the semen picture of cloned sires. Frozen semen from one clone bull was used for an AI trial, resulting in 65% pregnancies, 25 live calves were naturally delivered. Concerning the offspring of both female and male clones, the phenotypical and clinical observation of the calves in the first week of age did not reveal any clinical abnormality, suggesting that the deviations observed in clones are not transmitted to the progeny.

New strategies were proposed to improve the developmental competence of calf oocytes through in vitro technologies. Cumulus-oocyte complexes were first prematured for 24 h in the presence of meiosis inhibitors. Both Roscovitine alone (50 microM) or in combination with Butyrolactone-I (12.5 microM Rosco+6.25 microM BL-I) prevented the progression of meiosis. Their effect on nuclear maturation was reversible after a further 17 or 24 h maturation step. However, a dramatic decrease in embryo development was observed after fertilization (abattoir oocytes: 4-9% blastocyst rate versus 14-17% for control embryos). Similar results were obtained with oocytes collected by Ovum Pick Up from living donors. No pregnancy was obtained after single transfer of two blastocysts obtained from prematured oocytes (0/2 versus 4/12 for control embryos). Adding low concentrations (1, 3 or 10 microM) of follicular fluid-meiosis activating sterol (FF-MAS) during the maturation step had a beneficial effect on nuclear maturation (73-86% metaphase II versus 58% for control oocytes). However, subsequent embryo development was not improved. Enriching the maturation medium, namely with hormones, growth factors and precursors of glutathione, induced a sixfold increase in glutathione in the oocyte and had a beneficial effect on embryo development (38% increase in blastocyst rate). In conclusion, in opposition to the results reported with adult oocytes, prematuring calf oocytes had a negative impact on their developmental potential. Although FF-MAS improved nuclear maturation, its addition in the maturation medium did not increase embryo development. However, enriching the maturation medium had a positive effect on embryo development, indicating that cytoplasmic maturation was improved.