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Glutathione (GSH) plays a critical role in the elimination of reactive oxygen species (ROS) by acting as substrate for GSH peroxidase (GPX) which results in the oxidation of GSH. Glutathione reductase (GSR) regenerates GSH from oxidized glutathione (GSSG). In bovine oocytes, GSH concentration increases and is thought to have beneficial effects on embryo development. The goal of the present study was to analyze the GSH content in bovine blastocysts during blastocoel expansion and to determine the effects of tertiary-butyl hydroperoxide (tBH) in causing oxidative stress. It is metabolized by GPX with conversion of GSH to GSSG. Blastocysts were generated with standard protocols of in vitro production (IVP) using SOF medium supplemented with FAF-BSA and were collected at Days 7/8 of culture (Day 0 = IVF). Diameters of blastocysts were measured and embryos were allocated to 4 groups: early blastocysts (ebla, less than 135 �m), blastocysts (bla, 135–180 �m), expanded blastocysts (exbla, greater than 180 �m), and hatched blastocysts (hbla). Pools of the different categories (3 replicates, 36 embryos in total) were frozen at -80�C until assayed for GSH by HPLC. Total cell numbers were determined using the Hoechst 33342 stain. Blastocysts from the 4 groups were cultured together for 30 min in tBH (20 �M) containing SOF medium to induce oxidative stress. This was followed by another culture period for 1 or 8 h. Samples of blastocysts were frozen at -80�C at 0 h, 30 min, 1 h, and 8 h to assess GSH levels (Cereser et al. 2001 J. Chromatogr. B. Biomed. Sci. Appl. 752, 123–132) and transcript abundance. Semi-quantitative real-time RT-PCR was used to determine the relative abundance (RA) of GPX and GSR transcripts. Differences between groups were tested using ANOVA followed by a Tukey or Student's test (P ≤ 0.05). The grade of expansion significantly increased GSH concentration in bovine blastocysts. Expanded blastocysts (0.38 ± 0.04 ng) and hatched blastocysts (0.46 ± 0.05 ng) had a higher GSH content than early blastocysts (0.21 ± 0.01 ng) and blastocysts (0.27 ± 0.02 ng). However, GSH concentration remained constant in blastomeres during expansion and hatching as the number of cells increased from 54.9 ± 3.5 (ebla) to 152.3 ± 4.7 (hbla). After induction of oxidative stress, blastocysts showed a significant decrease in GSH content after a 1-h recovery period, followed by an increase in their GSH content after a period of 8 h (0 h: 0.26 ± 0.01 ng; 30 min: 0.25 ± 0.01 ng; 1 h: 0.20 ± 0.01 ng; 8 h: 0.32 ± 0.01 ng, respectively). This was accompanied by a significant increase of the RA of GSR transcripts in blastocysts collected after the 8-h recovery period compared to embryos at the other time points of the treatment. GPX mRNA abundance remained stable during the entire culture period. In the present study, GSH concentration was determined in bovine embryos during blastocoel expansion for the first time. The increased GSH content might to some extent be due to de novo GSH synthesis. GSSG is reduced to GSH most likely by GSR. Results show that bovine IVP embryos may serve as a new model for the study of oxidative stress.

RNA interference (RNAi) is now widely used for gene silencing in various biological systems. Injection of long double-stranded RNAs has been shown to specifically knock down gene expression in mammalian embryos. The utilization of short interfering RNAs (siRNA) to target specific embryonic genes would make this approach flexible and efficient enough for studying physiological functions in development. To demonstrate the feasibility of a single class of siRNA molecules for achieving long-lasting effects after injection into mammalian zygotes, we used siRNAs to knock down expression of the green fluorescent protein (GFP) in transgenic murine embryos of the OG2 transgenic line. Homozygous OG2-animals, carrying the Oct4-GFP transgene, were mated with NMRI animals to produce OG2 hemizygous zygotes, which show a parentally dependent expression pattern of the marker gene. Hemizygous zygotes with a maternally inherited Oct4-GFP gene continuously express the GFP marker; hemizygous zygotes with a paternally inherited Oct4-GFP start transcription of the GFP gene at the 4–8 cell stage, i.e., after onset of embryonic genomic activation. Thus efficacy and duration of gene silencing could be tested under 2 different conditions where (i) GFP mRNA was already present at the time point of injection, and (ii) GFP transcription started 2–3 cell cycles after siRNA injection. Zygotes were microinjected with either a 22-basepair GFP-siRNA or a control siRNA and then cultured in vitro. The siRNAs were conjugated with the fluorochome rhodamine to allow monitoring of injection and subsequent degradation of siRNAs. At the end of the in vitro culture, the developmental stage, the number of nuclei, GFP fluorescence (Table 1), and the GFP mRNA levels were determined by RT-PCR. In conclusion, results demonstrate that injection of a synthetic siRNA is sufficient to knock down a target gene transcript with either maternal or embryonic expression in mammalian embryos. Importantly, maternally derived GFP proteins showed a delayed functional knockdown of GFP for 2 days. The advantages of this approach are that (i) off-target effects of long double-stranded RNAs can be avoided, (ii) siRNAs against any known transcript can be rapidly designed and synthesized, and (iii) developmentally important genes can be silenced in embryos for at least 5 days following injection. Table 1.siRNA microinjection into zygotes with maternally and paternally inherited GFP

The first transgenic livestock species were reported in 1985. Since then microinjection of foreign DNA into pronuclei of zygotes has been the method of choice. It is now being replaced by more efficient protocols based on somatic nuclear transfer that also permit targeted genetic modifications. Lentiviral vectors and small interfering ribonucleic acid (siRNA) technology are also becoming important tools for transgenesis. In 2006 the European Medicines Agency (EMEA) gave green light for the commercialistion of the first recombinant protein produced in the milk of transgenic animals. Recombinant antithrombin III will be launched as ATryn for prophylactic treatment of patients with congenital antithrombin deficiency. This important milestone will boost the research activities in farm animal transgenesis. Recent developments in transgenic techniques of farm animals are discussed in this review.

Oocytes from prepubertal calves have a decreased developmental competence compared with oocytes from adult animals. The goal of this study was to improve the developmental competence of juvenile oocytes by maturation on granulosa cell (GC) monolayers from adult animals. Oocytes were recovered by ovum pickup (OPU) from 48 Holstein Friesian calves at 7-8 months of age and 18 adult cows. Animals received intramuscular injections of 60 mg FSH 48 h prior to each OPU session. Follicles were punctured twice per week in six consecutive OPU sessions. Cumulus oocyte complexes (COCs) recovered from calves were divided into three quality groups (classes I-III) and were then randomly distributed into three maturation groups: COCs were matured for 24 h on either GC or fibroblasts or without co-culture. Cow oocytes were matured without co-culture. TCM-199 supplemented with BSA (0.1%), hCG (5 IU/mL), and eCG (10 IU/mL) served as the medium in all groups. After maturation, all COCs were fertilized in vitro; after 18 h, presumptive zygotes were cultured in SOF+BSA for 8 days (37�C, 5% CO2). On Day 3, cleavage rates and, on Day 8, blastocyst rates were determined. The relative mRNA abundance of the following transcripts, critically involved in early embryonic development was determined: growth differentiation factor-9 (GDF-9), heat shock protein 70 (Hsp-70), and glucose transporter-3 (Glut-3). Single immature and matured oocytes (for GDF-9 and Hsp-70) and 8-16-cell embryos and expanded blastocysts (for Hsp-70 and Glut-3) from calves and cows were examined by semiquantitative RT-PCR. Cleavage and blastocyst rates were similar in oocytes derived from cows and calves matured on GC (74.3% vs. 70.0% and 22.3% vs. 22.3%, respectively), but were significantly higher (P < 0.05; one way ANOVA, Student-Newman-Keuls Method) than in the group without co-culture on fibroblasts (55.2% vs. 53.6% and 11.7% vs. 5.5%, respectively). GDF-9 expression was similar in immature calf and cow oocytes. After maturation, a significant decrease in GDF-9 expression was observed in calf oocytes. Matured cow oocytes showed a significantly higher mRNA abundance of GDF-9 than matured calf oocytes. The relative abundance of Hsp-70 was decreased in matured oocytes of all groups. Expanded blastocysts derived from adult oocytes expressed Hsp-70 significantly higher than blastocysts derived from oocytes of the control calves. The relative abundance of Glut-3 mRNA was similar in 8-16 cell embryos and expanded blastocysts in all groups. Overall, mRNA expression pattern for Hsp-70 and Glut-3 in blastocysts from GC matured oocytes were similar to that of cow blastocysts. Results indicate that maturation of juvenile calf oocytes on granulosa cells from adult animals improves their developmental competence. These findings provide clues toward identification of factors critically involved in acquiring full developmental capacity at puberty.

The effects of in vitro production (IVP) and/or somatic nuclear transfer on mRNA expression patterns have mostly been determined in morulae and blastocysts, i.e. after embryonic genome activation. Comparative data regarding mRNA expression patterns throughout the oviductal phase of pre-implantation development are scarce. Here we studied mRNA expression for genes related to DNA methylation and modification of histones which account for the major epigenetic reprogramming during development. Pertubated epigenetic reprogramming of the genome is a likely cause of developmental abnormalities and epigenetic diseases associated with assisted reproduction technologies. The objective of the present study was to compare mRNA expression of DNA methyltransferases Dnmt1, -3a, and -3b and histone methyltransferases SUV39-h1 and G9a between in vivo-derived bovine embryos and their IVP counterparts using a semiquantitative RT-PCR assay (Wrenzycki et al. 2002 Biol. Reprod. 66, 127-134) employing two embryos for each assay. In vivo-derived embryos were collected from 28 superovulated heifers by endoscopic flushing of oviducts (zygotes to 8-cell stages) (Besenfelder et al. 2001 Theriogenology 55, 837-845) or by uterine flushing (16-cell stages to blastocysts). Endoscopic flushing at different time points after AI (Days 1, 1.5, 2, 3, 4, and 4.5) yielded 31 zygotes; 15 two-cell, 5 three-cell, 13 four-cell, 1 five-cell, 2 six-cell, and 11 eight-cell embryos; 4 degenerated embryos; and 18 unfertilized ova. The recovery rate (corpora lutea counted per recovered embryos) was 58% and 62% for the endoscopic and uterine flushing, respectively. Differences in the relative abundance of each gene transcript between groups were tested using ANOVA with the main effects being origin (in vivo/in vitro) and developmental stage (zygote to blastocyst) and their interactions followed by multiple pairwise comparisons using a Tukey test (P < 0.05). Origin of embryos affected the relative abundance of transcripts for Dnmt1, Dnmt3a, and SUV39-h1, and developmental stage affected the relative abundance of transcripts for Dnmt1, -3a, -3b, SUV39-h1, and G9a. No interactive effects were observed for origin and developmental stage in the relative abundance of all transcripts. The relative abundance of Dnmt1 transcripts differed significantly between in vivo- and in vitro-produced morulae and blastocysts. For Dnmt3a, mRNA differences were determined between in vivo- and in vitro-produced 10-16-cell stages and morulae. Suv39-h1 transcripts differed significantly between in vivo- and in vitro-derived zygotes, 2-cell embryos, 8-cell embryos, 10-16-cell embryos, and blastocysts. The results suggest that IVP alters mRNA expression of genes related to epigenetic modifications very early in development, even before the embryonic genome has been activated.

The effects of the cytosceletal inhibitor cytochalasin in solid surface vitrification (SSV; Dinnyes et al. 2000 Biol. Reprod. 63, 513–518) were investigated on in vitro matured (IVM) porcine oocytes. Cumulus-free IVM oocytes were subjected to one of the following: SSV (equilibration in 4% ethylene glycol (EG) for 10 min followed by vitrification in 35% EG, 5% polyvinyl pirrolidone, and 0.3 M trehalose on a cold (about −150°C) surface; warming in 0.4 M trehalose at 37°C), SSV pre-treatment with 5 μg/mL cytochalasin B (SSV + CB), or the steps of SSV without cooling, i.e. toxicity control (TC). Non-lysed oocytes together with the non-treated controls were subjected to parthenogenetic activation and then in vitro cultured (IVC) for six days. The proportion of non-lysed oocytes was higher when pre-treatment with CB was performed compared to SSV. However, both results were significantly lower than that of the TC. After parthenogenetic activation via a combination of a direct current electric pulse (1 kV/cm for 45 μs) followed by 3 h treatment with 2 mM 6-dimethylaminopurine, the proportion of cleaved embryos was higher in the SSV + CB than in the SSV. Nevertheless, significantly more oocytes cleaved in the TC and control groups. On Day 6 no blastocyst, were determined in the SSV group, and one blastocyst was obtained in the SSV + CB, while significantly more blastocysts developed in both the TC and the control (Table 1). There was no significant difference in blastocyst rates and in the number of blastomere nuclei/embryo between the TC and the control. These results indicate that the high concentration of cryoprotectants per se applied in SSV were not detrimental for in vitro development and that CB pre-treatment increased survival and further development of SSV vitrified pig oocytes resulting in one parthenogenetic blastocyst from vitrified pig oocytes. Table 1. Parthenogenetic development of vitrified IVM porcine oocytes This research was supported by a Bilateral Scientific and Technological Collaboration (TET, No. D6/01) grant between Germany and Hungary and by the Hungarian National Office of Research and Technology (OM-KMUFA; BIO-00086/2002).

In vitro production (IVP) of porcine embryos by in vitro maturation of oocytes followed by fertilization and culture in vitro is hampered by great deficiencies. Initiation of at least the major embryonic genome transcription, which includes activation of ribosomal RNA (rRNA) genes and the associated formation of a fibrillo-granular nuclealus, is normally seen during the 4-cell stage in pigs. We have investigated the activation of rRNA synthesis and the presence of silver staining nucleolar proteins in porcine IVP embryos as a marker of transcriptional activity and, thus, developmental competence. A total of 205 porcine IVP embryos from the 2-cell to the blastocyst stage were examined using sequential fluorescent in situ hybridization (FISH) to the rRNA genes and their transcripts and silver staining of nucleolar proteins as previously described (Viuff et al. 2002 Biol. Reprod. 66, 629–634). Briefly, cumulus-oocyte complexes with at least three cumulus cell layers and evenly granulated ooplasm were isolated from 2–5 mm ovarian follicles with stereomicroscopic evaluation. Subsequently, oocytes were matured in NCSU-37 and mechanically denuded followed by fertilization using frozen-thawed epididymal semen. Presumptive zygotes were then cultured in NCSU-23 at 39°C, 5% CO2. Around the time of expected cleavage, the embryos were examined every second hour to determine the time of cleavage. Embryos at the 2-cell stage were harvested at 5 h post-cleavage (hpc), 4-cell embryos late during the third cell cycle at 30 hpc, and tentative 8- and 16-cell embryos at 10 hpc. Blastocysts were harvested at Day 5 post-insemination. In general, nuclei of 2-cell embryos displayed 4 small foci of FITC labelling (presumably the rDNA), but no specific silver staining, and were consequently categorized as transcriptionally inactive. At the late 4-cell stage, 58% of the embryos resembled the 2-cell stage. However, in the remaining embryos (42%), some or all nuclei displayed large areas of FISH labelling (presumptive rDNA and rRNA) co-localized with silver staining, and were catagorized as transcriptionally active. Among the 8-cell embryos, 64% displayed a majority of transcriptionally active nuclei, whereas this was the case in 83% and 92% of the embryos in the 16-cell embryos and the blastocysts, respectively. In general, the majority of the embryos contained a mixture of transcriptionally active and inactive cells. These findings show that the porcine IVP embryos are often delayed and asynchronous with respect to activation of the rRNA genes. Table 1. Categorization of nuclei according to transcriptional activity This work was supported by grants from “Disease models, disease prevention and animal welfare improvement: The pig embryo as a model.” Danish Research Agency (Grant: 9901178), NATO (Grant: 978658), and Deutsche Forschungsgemeinschaft (DFG).

In mammals, DNA methylation and the modification of histones account for the major epigenetic alterations. Usually DNA methylation is associated with transcriptional silencing, which is catalyzed by two important classes of DNA methyltransferases. DNA methyltransferase 1 (Dnmt1), a maintenance enzyme, methylates hemi-methylated DNA after DNA replication. Dnmt3a and Dnmt3b are required for de novo methylation in vivo and for establishing new DNA methylation marks during development. In addition to DNA methylation, post-translational modifications of the amino termini of core histones are thought to affect the expression or repression of transcription. Histone deacetylation catalyzsed by histone deacetylases (Hdac) often results in transcriptional repression. Perturbated epigenetic reprogramming of the genome is a likely cause of developmental abnormalities and epigenetic diseases introduced by assisted reproduction technologies. The objective of the present study was to determine the relative abundance of Dnmt1, Dnmt3a, and Hdac2 transcripts in ICM and TE cells of pre-implantation bovine embryos of different origin. Embryos were generated with standard protocols of in vitro production (IVP) and parthenogenetic activation using SOF medium supplemented with BSA (SOF d7/d8, Parth d7/d8) or TCM medium supplemented with estrus cow serum (TCM d7/d8; Wrenzycki et al. 2001 Hum. Reprod. 16, 893–901; Wrenzycki et al. 2002 Biol. Reprod. 66, 127–134). Expanded blastocysts were collected at Days 7 and Day 8 (d7/d8) of culture (Day 0 = IVF). In vivo-generated blastocysts collected from superovulated animals were included as controls (Wrenzycki et al. 1996 J. Reprod. Fert. 108, 17–24). A highly sensitive RT-PCR assay (Wrenzycki et al. 2003 Biol. Reprod. 68, 2073–2080) was used to determine the relative abundance (RA) of gene transcripts in isolated ICM and TE cells. The allocation of ICM and TE cells was analyzed using a differential staining technique (Eckert and Niemann 1998 Mol. Hum. Reprod. 4, 957–965) to calculate the RA of each transcript on a per cell basis. RT-PCR assays were repeated at least six times. Significant differences in the RA of Dnmt1 (In vivo, TCM d7, Parth d7/d8, SOF d8), Dnmt3a (In vivo, SOF d7), and Hdac2 (TCM d7) transcripts were detected between ICM and TE cells. No differences were detected for all transcripts in ICM cells of embryos collected at either Day 7 or Day 8. In TE cells, the RA of Dnmt1 transcripts was significantly reduced or increased in embryos generated in SOF or TCM medium at Day 7, respectively, and the RA of Hdac2 transcripts was significantly higher in embryos generated in TCM medium at Day 8. These results suggest that in vitro culture alters the spatial expression pattern of genes related to epigenetic modifications in the cell lineages critical for a normal embryonic and fetal development. This work was supported by the Deutsche Forschungsgemeinschaft (DFG Ni 256/18-1).

Porcine nuclear transfer (NT) is an inefficient process and it is necessary to use as many as 120 NT embryos for each foster mother to obtain small litters of live piglets. In these experiments, we evaluated the effects of culture atmosphere and medium on the development of NT embryos by monitoring blastocyst rate and cell number of Day 6 blastocysts. Age matched IVF and parthenogenetic embryos were also evaluated for comparison. For all experiments a pool of oocytes was aspirated from ovaries collected in a local abattoir. Following aspiration, oocytes were allowed to mature for 40 h in North Carolina State University (NCSU)-37 medium (supplemented with cAMP and hCG/eCG for the first 22 h). After removal of the cumulus cells, denuded oocytes with polar bodies were selected for NT, enucleated, fused with fetal fibroblasts, and sequentially activated electrically and chemically by 3 h of treatment with 6-dimethylaminopurine (6-DMAP). A second group of oocytes from the same denuded pool were maintained in TL-HEPES medium and activated in parallel with the NT group to produce parthenogenetic embryos. A third group was fertilized with frozen-thawed epididymal semen and co-cultured for ∼12 h to give IVF embryos. All three treatment groups were subdivided into a control subgroup and an experimental subgroup. In the first experiment, we compared the effects of atmosphere (20% vs. 5% oxygen) on in vitro embryonic development in NCSU-23 medium. In the second experiment, we used only the 5% oxygen concentration and compared different culture media. One subgroup was maintained in standard NCSU-23 medium and the second subgroup was cultured in a two-step system for the first 58 h in modified NCSU-23 (without glucose but supplemented with 2.0 mM lactate and 0.2 mM pyruvate), followed by addition of glucose to give a final concentration of 5.55 mM. Data were statistically analyzed by analysis of variance and chi square test. Blastocyst rate and mean cell number in all three embryo groups were improved under 5% oxygen. The most dramatic effect was observed in the NT group, in which the blastocyst rate increased significantly (P < 0.001) from 6.7% ± 5.9 (n = 279) to 19.6% ± 8.9 (n = 250) and mean cell number increased from 17.7 ± 12.1 to 25.8 ± 10.3 cells per blastocyst. With 5% oxygen there was also an increase of blastocyst rates and mean cell numbers in both IVF and parthenogenetic groups. In the second experiment, blastocyst rate for NT embryos increased significantly (P < 0.05) from 21.8% ± 7.6 (n = 242) in conventional NCSU-23 to 31.5% ± 11.0 (n = 271) in the modified system whereas there was almost no difference in the mean cell number of both groups (29.2 ± 4.3 vs. 31.5 ± 5.3). In the groups of IVF and parthenogenetic embryos no difference was found. These results indicate that both the reduced oxygen and the modified culture medium are important for pre-implantation development of porcine nuclear transfer embryos.

Array technology is a widely used tool for gene expression profiling, providing the possibility to monitor expression levels of an unlimited number of genes in various biological systems including preimplantation embryos. The objective of the present study was to develop and validate a bovine cDNA array and to compare expression profiles of embryos derived from different origins. A bovine blastocyst cDNA library was generated. Poly(A+)RNA was extracted from in vitro-produced embryos using a Dynabead mRNA purification kit. First-strand synthesis was performed with SacIT21 primer followed by randomly primed second-strand synthesis with a DOP primer mix (Roche) and a global PCR with 35 cycles using SacIT21 and DOP primers. Complementary DNA fragments from 300 to 1500bp were extracted from the gel and normalized via reassoziation and hydroxyapatite chromatography. Resulting cDNAs were digested with SacI and XhoI, ligated into a pBKs vector, and transfected into competent bacteria (Stratagene). After blue/white colony selection, plasmids were extracted and the inserts were subjected to PCR using vector specific primers. Average insert size was determined by size idenfication on agarose gels stained with ethidium bromide. After purification via precipitation and denaturation, 192 cDNA probes were double-spotted onto a nylon membrane and bound to the membrane by UV cross linking. Amplified RNA (aRNA) probes from pools of three or single blastocysts were generated as described recently (Brambrink et al., 2002 BioTechniques, 33, 3–9) and hybridized to the membranes. Expression profiles of in vitro-produced blastocysts cultured in either SOF plus BSA or TCM plus serum were compared with those of diploid parthenogenetic ones generated by chemical activation. Thirty-three probes have been sequenced and, after comparison with public data bases, 26 were identified as cDNAs or genes. Twelve out of 192 (6%) seem to be differentially expressed within the three groups;; 7/12 (58%) were down-regulated, 3/12 (25%) were up-regulated in SOF-derived embryos, and 2/12 (20%) were up-regulated in parthenogenetic blastocysts compared to their in vitro-generated counterparts. Three of these genes involved in calcium signaling (calmodulin, calreticulin) and regulation of actin cytoskeleton (destrin) were validated by semi-quantitative RT-PCR (Wrenzycki et al., 2001 Biol. Reprod. 65, 309–317) employing poly(A+) RNA from a single blastocyst as starting material. No differences were detected in the relative abundance of the analysed gene transcripts within the different groups. These findings were confirmed employing the aRNA used for hybridization in RT-PCR and showed a good representativity of the selected transcripts. Results indicate that it is possible to construct a homologous cDNA array which could be used for gene expression profiling of bovine preimplantation embryos. Supported by the Deutsche Forschungsgemeinschaft (DFG Ni 256/18-1).

In spite of in vitro embryo production systems having been greatly improved over recent years, employing a variety of culture conditions (media, protein sources, gas atmosphere, etc.), we still do not know much about the real necessity of embryos to develop under the same conditions as occur in vivo. These differences between in vivo and in vitro culture at preimplantation embryonic stages can produce deviations in gene expression and in normal fetal development (large offspring syndrome). Heat shock proteins (Hsp) are engaged in cell response to regulatory signals or perturbations in the microenviroment and can be used as a sensitive indicator of stress caused by suboptimal culture conditions (Wrenzycki et al., 2001Hum. Reprod. 16, 893–901). Hsp act as chaperones in facilitating protein folding and assembly and stabilize damaged proteins to prevent aggregation of fragments, thereby allowing repair or degradation. The aim of the present study was to investigate the effects of different embryo/volume ratios on bovine embryo development and the relative abundance of Hsp 70.1 gene transcripts. In this experiment, oocytes were isolated from slaugterhouse ovaries and matured, fertilized and cultured in groups of 5, 10, 20 or 30 per each drop of 100μL. The oocytes were matured in TCM 199 supplemented with 0.4% BSA. After maturation, oocytes were fertilized in TALP medium, using frozen/thawed sperm, selected using a percoll density gradient. The zygotes were cultured to the morula or Day 7 blastocyst stage employing SOF supplemented with 0.4 % BSA. Developmental check points were cleavage rate (Day 3pi), blastocyst formation (Day 8pi) and hatching (Day 11pi). A semi-quantitative RT-PCR assay was used to determine the relative levels of gene transcripts in single embryos at morula (Day 6) and blastocyst (Day 7) stages (Wrenzycki et al., 2001 Biol. Reprod. 65, 309–317). Data of cleavage, blastocyst formation and hatching rates were analyzed using chi-square test. Relative abundance (RA) of Hsp 70.1mRNA were compared in tested groups using ANOVA followed a Tukey test. Differences at P

Several imprinted genes have been identified in different species such as mouse and man but in cattle imprinting has only been confirmed for the Insulin-like growth factor 2 receptor gene (Igf2r). DNA methylation is one of the most common mechanisms of imprinting. Imprinting is correlated with the methylation of normally unmethylated CpG islands, and aberrations in methylation are thought to be involved in the Large Offspring Syndrome (LOS) which is frequently observed in offspring derived from in vitro-produced and/or cloned embryos. The imprinting of the bovine Insulin-like growth factor 2 gene (Igf2), the Igf2r gene and the Mammalian achaete scute homologue 2 gene (Mash2) was analyzed in bovine preimplantation blastocysts cultured in SOF+BSA. mRNA levels of the Igf2r gene and the Mash2 gene in in vitro-produced (IVP) and parthenogenetic expanded single blastocysts were analyzed by semi-quantitative RT-PCR (Wrenzycki C et al., 2001 Biol. Reprod. 65, 309–317). Genes expressed exclusively from the maternal allele (i.e. paternally methylated) should be represented by a higher relative abundance of transcriptional products in parthenogenetic embryos whereas paternally expressed genes (i.e. maternally methylated) should be correlated with a higher gene expression in IVP embryos carrying one paternal and one maternal allele. For determination of methylation patterns by bisulfite sequencing, sequences of several DNA fragments from the bovine Igf2 gene were identified in samples from bovine uterus and kidney. The primer pairs were generated from the ovine and bovine Igf2 sequences available in the Genebank database (accession number U00664/X53553). DNA fragments identified were from the 5′ untranslated region and the 3′ translated region of the bovine Igf2 gene. All fragments consist of a high number of CG dinucleotides, and computer analysis using the CpGwin program (Anbazhagan R et al., 2001 BioTechniques 30, 110–114) revealed CpG islands within these fragments. Relative abundances of transcriptional products were statistically analyzed using the SigmaStat 2.0 (Jandel Scientific, San Rafael, CA, USA) software package. After testing for normality, an ANOVA followed by multiple pairwise comparisons using the Tukey test was employed. Results from the semi-quantitative RT-PCR analyses revealed no difference (P>0.05) in gene expression pattern of the Igf2r gene, suggesting that the Igf2r gene is biallelically expressed during bovine preimplantation development. In contrast, there was a significant difference (P

The role of exogenous protein during bovine pre-implantation embryo development in two in vitro production systems was investigated. Morphological development, survival after vitrification and metabolic activity before and after vitrification were recorded in blastocysts generated in vitro in synthetic oviduct fluid (SOF) medium in the presence of either bovine serum albumin (BSA) or polyvinyl-alcohol (PVA). Metabolic activity was determined by measuring oxygen consumption, glucose and pyruvate uptake as well as lactate production. Development to blastocysts and survival after vitrification were reduced significantly in medium lacking protein. Of the metabolic parameters measured, only pyruvate uptake was increased significantly in embryos cultured in medium supplemented with PVA. Whereas in BSA-supplemented medium pyruvate uptake was correlated with lactate production, in PVA-supplemented medium glucose uptake was correlated with lactate production. Lactate production increased significantly after vitrification as compared with fresh embryos. Thus, exogenously added protein significantly alters oxidative metabolism. In medium lacking protein, the additional pyruvate may be used for the maintenance of intracellular amino acid pools. Vitrification appears to alter glycolytic metabolic profiles indicating a stress-response. In conclusion, the perturbed metabolism corresponding to reduced developmental capacity of embryos produced under protein-free conditions emphasizes the ambiguity between maximum develop-ment, technical and hygienic requirements and physiological demands of the early bovine embryo in vitro. The use of well-defined recombinant proteins might assist in closing this gap.