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In breeding programs, the application of a vitrification method suitable for direct transfer of biopsied embryos can increase the genetic improvement of cattle and help reduce the costs of embryo transfer. The aim of this study was to determine the in vitro survival of biopsied vitrified blastocysts using the new VitTrans device (Morató and Mogas 2014 Cryobiology 68, 288-293), a 1-step in-straw warming system. Immature bovine oocytes were in vitro matured, fertilized, and cultured to the blastocyst stage. A total of 110 grade 1 blastocysts (IETS codes 6 and 7) were randomly allocated to 2 groups: (1) biopsy (n=49) and (2) without biopsy, or control (n=61). Blastocysts were biopsied using a microblade mounted on a micromanipulator. A small portion of the trophoblast, approximately 15%, was cut off and a significant part of the zona pellucida was sliced away. Both groups were then vitrified using the VitTrans device. For vitrification, all blastocysts were exposed to an equilibration medium with 7.5% ethylene glycol+7.5% dimethyl sulfoxide in holding medium (HM) consisting of TCM-199 with 20% FCS, moved into a drop with 16.5% ethylene glycol+16.5% dimethyl sulfoxide+0.5M sucrose in HM, and then placed in a microdroplet on the VitTrans. The VitTrans was plunged into LN and covered with a 0.5-mL straw. For warming, the protective cover was removed from the VitTrans while still submerged in LN. Subsequently, a new 0.5-mL plastic embryo transfer straw was placed on the VitTrans while flushing the warming solution (0.3mL of 0.5M sucrose in HM at 45°C) with a syringe through the lumen of the device. By entering the warming solution into the VitTrans device, the embryo is flushed inside the plastic straw. The straw containing the embryo can then be readily used for transfer after the VitTrans is removed. To recover the embryo in the laboratory, the content of the straw was put into a Petri dish and blastocysts were placed in the culture medium and incubated at 38.5°C in 5% CO2 and 5% O2 in air. Morphology and re-expansion were evaluated 24h post-warming. The embryo survival rate was defined as the ratio of blastocysts that were able to re-expand with regards to the total number of warmed blastocysts. Due to the attachment of embryos inside the straw, a total of 18 embryos were lost during recovery (12 from the biopsied group and 6 from the nonbiopsied group). The ratio of re-expanded blastocysts from the recovered embryos was 40% in the biopsy group and 61% in the control group. In conclusion, vitrification using the VitTrans device showed good results with intact embryos compared with biopsied embryos. In addition, biopsied embryos had a tendency to adhere to the inside of the straw, which is probably due to the damage or loss of the zona pellucida. Additional research is required to minimize the loss of embryos.

We generated a CRISPR/Cas9 mediated knockout (KO) of the OCT4 gene in adult fibroblasts, where biallelic deletion of a single nucleotide leads to a frameshift mutation. Through reconstruction of embryos by somatic cell NT, we were able to study the role of OCT4 during pre-implantation development until Day 7 in vitro. The presence of OCT4 protein was evaluated after immunofluorescent staining by confocal laser scanning microscopy of Day 5 morulae and Day 7 blastocysts; somatic cell NT embryos reconstructed from nontransfected cells of the same source served as control. Whereas control morulae expressed OCT4 in all cells, OCT4 KO morulae showed expression in only 67.8 ± 11.1% (mean ± SD, n = 6) of cells and overall intensity was decreased. By Day 7, no expression of OCT4 was detected in OCT4 KO blastocysts (n = 24), suggesting that maternal stores of the OCT4 protein had decayed. In contrast, control blastocysts (n = 20) showed OCT4 expression ubiquitously in both inner cell mass (ICM) and trophectoderm (TE). Simultaneously to the OCT4 staining, we differentially stained ICM and TE with the TE specific marker CDX2 and counterstained cell nuclei with 4′,6-diamidino-2-phenylindole. No significant differences between OCT4 KO Day 7 blastocysts and controls were detected in total cell numbers (89.6 ± 27.5 v. 96.3 ± 38) and percentage of CDX2 positive cells (50.7 ± 16.8% v. 59.0 ± 20.8%) (P > 0.05, mean ± SD, unpaired, two-tailed t-test). To analyse the role of OCT4 during the second lineage differentiation, we stained Day 5 morulae and Day 7 blastocysts for the epiblast and hypoblast specific markers NANOG and GATA6, respectively. In morulae, both markers were present and co-expressed in OCT4 KO and control embryos. By Day 7, control blastocysts (n = 6) already showed the typical salt and pepper distribution of NANOG and GATA6 positive cells, but expression was not mutually exclusive in all cells and also not restricted to ICM. OCT4 KO embryos lost all NANOG expression at Day 7 blastocyst stage (n = 8) and only stained positive for GATA6 in both TE and ICM. We conclude that OCT4 is not required for the quantitative allocation of cells to either the ICM or the TE during the first lineage differentiation, as total cell number and percentage of CDX2 positive cells was unchanged. Additionally, expression of NANOG seems to be OCT4 dependent and OCT4 KO embryos fail to establish the epiblast lineage—unlike mouse Oct4 KO embryos, where developmental failure was connected to loss of GATA6 expression during second lineage differentiation. This work was funded by the Bavarian Research Foundation (AZ-1031–12).

Cattle can serve as a model organism to resolve central questions in mammalian reproduction that cannot be clarified in the mouse model due to notable species-specific peculiarities of early mouse embryogenesis. As part of a project on structural, molecular, and functional deficiencies of bovine oocytes, we started to systematically investigate fertilization and the onset of embryo development in vitro by 3-dimensional multicolor fluorescence microscopy. We are using 3D visualisation as key approach to clarify the multiple parallel and sequential processes and events of fertilization as well as to identify and classify errors and failures. Moreover, we aim to gain insights into the mechanisms of aberrations by linking processes at the cellular and the molecular level. We studied class I and II oocytes collected from slaughterhouse ovaries and matured for 23 h in vitro. Oocytes were fixed at different times from 4 to 12 h postinsemination with formaldehyde in a microtubule-stabilising buffer containing taxol in such a way that the 3-dimensional cell architecture was maintained, and were stained for DNA, microtubules, and f-actin microfilaments. In addition, serine 10-phosphorylated histone H3 was used as a marker for chromosome condensation and the spindle midbody. For 3-dimensional imaging of the oocytes in toto, confocal serial sections were captured at 1-µm intervals using a 40× objective (NA = 1.3). For imaging details, we used a high spatial sampling density (pixel size: 50 × 50 nm, z-step size: 200 nm) close to the Nyquist criterion and image restoration by maximum likelihood estimation (MLE) deconvolution. A series of more than 500 three-dimensional snapshots of fertilized oocytes at different points in time gives a first detailed view on the spatial and temporal course of the sperm entry, the formation of the paternal pronucleus and the sperm aster, completion of oocyte meiosis and the formation of the maternal pronucleus, as well as dynamic changes of the cytoskeleton. Moreover, we can document a spectrum of abnormalities including spontaneous parthenogenetic oocyte activation, polyspermy, and aberrations of meiosis I and II. The latter include irregular spindle formation and chromosome segregation, the occurrence of chromatin bridges and abnormal spindle positioning and rotation (e.g. leading to nonextrusion of a first or a second polar body or the extrusion of two second polar bodies). Our microscopic investigation in the bovine system contributes to unraveling the origins of irregular cleavage, aneuploidy, and mosaicism in mammals. Three-dimensional high-speed microscopy of oocytes and zygotes in affordable timeframes could be of great value in improving the differential diagnosis of oocyte and sperm dysfunction, as well as in identifying and dissecting problems, limitations, and potential risks of reproductive technologies (ART).This work is supported by the Deutsche Forschungsgemeinschaft (DFG FOR 1041).

The aim of this study was to develop a reliable procedure for genomic evaluation of bovine embryos to determine gender, polled status, and hereditary defects within 24 h after collection. German Simmental animals (n = 15) were superovulated (n = 25) using a standard protocol. Embryos were recovered on Day 7 (Day 0 = oestrus). A total of 217 embryos (morula, n = 130; early blastocyst, n = 43; blastocyst, n = 44) were biopsied with a steel blade attached to a micromanipulator. Biopsied cells were immediately transferred into 1 µL TE buffer to a 500 µL reaction tube and embryos were in vitro cultured until genomic results were available. For commonly used molecular genetic methods (e.g. 5′-exonuclease genotyping, PCR or high density genotyping) DNA amounts of 2–200 ng are required. However, the DNA quantity of a single diploid cell amounts to 6 pg only. The embryo biopsies used, usually consists of 10–30 cells, necessitating an artificial amplification of the embryonic genome. Taking all vital measures to avoid external DNA contamination, isothermal whole genome amplification was performed with the REPLI-g Mini Kit (Qiagen, Valencia, CA, USA) using random hexamers and Phi29-Polymerase. Depending on the number of cells, a total DNA amount of 4–7 µg was achieved. Polled status and gender was determined using PCR with subsequent gel-electrophoresis. 5′-exonuclease assays were used to obtain genotypes for the detection of genetic defects. At present, eight, mostly Simmental-specific genetic disorders can be examined: three traits associated with severe growth retardation, dwarfism (DW), Braunvieh-haplotype 2 (BH2) and stunted growth (FH2), the lethal skin disorder zinc deficiency-like syndrome (ZDL), a fertility trait bovine male subfertility (BMS), embryonic death Fleckvieh-haplotype 4 (FH4), a bleeding disorder thrombopathia (TP) and arachnomelia (A), within 24 h. On average, 8.7 embryos were biopsied per embryo recovery, i.e. 93.9% of the total number of transferable embryos. Fourteen embryo samples (6.5%) totally failed during analysis, possibly due to the loss of samples. In successful analyses, gender was undetermined in two embryos; remaining embryos were 52.2% female and 47.8% male. Polled status could be analysed in 92.6% of the embryos. The analyses of embryos for possible inherited genetic disorders (healthy, heterozygote, or homozygote; n = 578) were successful in 90.1%. The transfer of biopsied embryos (n = 30) led to 63.3% pregnancies (Day 42). A validation of the present results has to be done as soon as the produced calves are born, demonstrating the reliability of the procedure.Research was funded by the Bayerische Forschungsstiftung (AZ-1031-12).

As a part of a study on bovine embryo genomic evaluation, effects of an intact (iZP) or opened zonae pellucidae (oZP) of in vitro-produced embryos on vitrification outcomes were assessed. In the first experiment, only iZP embryos were subjected to vitrification, using either the hollow fibre vitrification method (HFV; Matsunari et al. 2012 J. Reprod. Dev.) or the cryologic vitrification method (CVM; CryoLogic®, Blackburn, Victoria, Australia). Developmental stage (morula = M; early blastocyst = EaB; blastocyst = B) and quality (good = 1; fair = 2) before vitrification were evaluated. In a first set of experiments, quality 1 and 2 iZP embryos were vitrified either by the HFV or the CVM method. A significant difference between the 2 methods was found when comparing overall survival rates (24–48 h post-thaw; HFV 59.32% v. CVM 78.9%; P 0.1). In order to evaluate influence of embryo stage and quality on HFV or CVM outcomes, iZP embryos vitrified by the HFV method were classified regarding quality (1: n = 207; 2: n = 66; total n = 273) and embryo stage (M: n = 78; EaB: n = 74; B: n = 121). Concerning survival rates, no significant difference was found between M 1 (57.4%) and M 2 (44.6%), or EaB 1 (80%) and EaB 2 (68%). However, a significant difference was found when comparing B 1 (77%) and B 2 (29%; P 0.1) was found. Effect of ZP, either intact or opened, does not seem to affect survival rates (judged by their re-expansion 24–48 h post-thaw) of good-quality embryos.The authors thank the Bavarian Research Foundation for the financial support (AZ-1031-1; DOK-153-12).

The aim of the present study was to develop a reliable method to simultaneously split and biopsy valuable bovine embryos for a complete genomic evaluation (gender, polledness, and hereditary abnormalities) and to estimate the breeding value of progeny for traits of economic importance immediately after embryo recovery. A total of 208 good quality embryos collected from superovulated German Simmental animals were biopsied immediately after recovery using an inverse microscope (Zeiss, Germany) at 50× magnification with a single-use steel blade mounted on a holder (Bausch & Lomb, Germany) attached to a micromanipulator (Eppendorf, Germany). Biopsy was performed either by splitting the embryo and cutting of one-third of a half [G1: morulae (M), n = 50; early blastocysts (EB), n = 24; blastocysts (B), n = 16], by just splitting in equal halves (G2: M, n = 16; B, n = 2), or by cutting of just a small biopsy of the embryo (G3: M, n = 53) or of the trophoblast (G3: EB, n = 19; B, n = 28). Biopsied cells were immediately used for DNA amplification. Biopsied embryos (E) and demi-embryos (DE) were in vitro cultured in SOF, under mineral oil, at 39°C and 5% CO2, 5% O2, 90% N2 for 24 h, after which survival was recorded. Survival rate of G1 (survival of at least 1 DE: M, 98.0%; EB, 100.0%; B, 93.8%), G2 (survival of DE: M, 75.0%; B, 100.0%), and G3 (embryo survival: M, 96.3%; EB, 100.0%; B, 96.4%) were similar, but in relation to the number of original embryo the highest ratio of DE was obtained in G1 (1.67) v. G2 (0.88) and G3 (0.97; G1:G2/G3; P

The multiplication of high-value embryos by chimera formation using asynchronic aggregation is a promising alternative to embryonic cell nuclear transfer. Single blastomeres from a donor embryo are aggregated with 2 host embryos, thus several chimeras can be constructed per donor embryo. Due to the advanced developmental stage, the donor blastomeres are likely to contribute to the inner cell mass (ICM) and later give rise to the embryo proper, whereas the host embryos form extra-embryonic tissues. To test if pairs of blastomeres from Day 5 morulae are able to form the ICM when aggregated with 2 Day 4 host embryos, we produced transgenic donor embryos carrying a fluorescent reporter gene (enhanced green fluorescent protein, eGFP) by using semen from an eGFP transgenic bull (Reichenbach et al. 2010 Transgenic Res. 19, 549–556) for in vitro fertilization and in vitro host embryos produced by a standard procedure. The zona pellucida of all embryos was removed by treatment with 1 mg mL–1 pronase. Donor embryos were assessed for eGFP expression by fluorescence microscopy and disaggregated by gentle pipetting after incubation in Mg2+- and Ca2+-free medium. Pairs of blastomeres were then placed between 2 host embryos and cultured individually in a well-of-the-well culture dish. On Day 6 after aggregation, fully developed blastocysts were assessed for eGFP fluorescence. In 3 replicates, n = 30 chimeras were produced by aggregation; 13 (43%) developed to blastocysts, of which 2 (15%) showed local eGFP expression in the ICM and 7 (54%) showed a generalized expression. From the results of this study we conclude that Day 5 morulae may be multiplied in an efficient manner by using the chimera formation technique, which makes this approach applicable to ex vivo-derived embryos. In future investigations we will study the effect of using donor blastomeres from either the inside or outside of the donor morula and test the use of tetraploid host embryos to increase the rate of blastocysts with the desired genotype in the ICM. Finally, we aim to introduce this multiplication approach to the production of genotyped embryos with a genomic estimated breeding value (gEBV) and intend to produce calves with identical gEBV.Funded by the Bavarian Research Foundation (AZ-1031–1).

Three-dimensional (3-D) super-resolution fluorescence microscopy has allowed major progress in studies of the functional nuclear organization (Markaki et al. 2010 Cold Spring Harb. Symp. Quant. Biol. 75, 475–492; Markaki et al. 2012 Bioessays 34, 412–426). We have exploited these new possibilities to explore nuclear organization at different stages of bovine pre-implantation development (4-cell, 8-cell, 16-cell, morula, and blastocyst stage). In particular, we studied the topography of RNA polymerase II and the distribution of transcriptionally competent and noncompetent chromatin using antibodies against H3K4me3 and H3K27me3, respectively. For comparison, we have started analyses of mouse pre-implantation embryos and embryonic stem cells as well. Our results support the chromosome territory-interchromatin compartment (CT-IC) model (Cremer and Cremer 2010 Cold Spring Harb. Perspect. Biol. 2, a003889; Cremer et al. 2012 In: Epigenetic Regulation and Epigenomics 451–483). In all cell types, the nuclear space is occupied by chromosome territories (CTs; Koehler et al. 2009 Exp. Cell Res. 315, 2053–2063), the interchromatin compartment (IC), and one or several nucleoli. The CTs are built up from interconnected, megabase-sized chromatin domains (CDs). These ~1-Mbp CDs may consist of a series of ~100-kbp CDs (Cremer et al. 2000 Crit. Rev. Eukaryot. Gene Expr. 10, 179–212), which globally form a compact chromatin core surrounded by a layer of decondensed chromatin, called the perichromatin region. Current evidence supports the hypothesis that the perichromatin region represents the nuclear compartment, where transcription, co-transcriptional splicing, DNA-replication, and DNA-repair take place (Rouquette et al. 2010 Int. Rev. Cell Mol. Biol. 282, 1–90). The IC provides a contiguous, crowded compartment, which starts with channels at nuclear pores and pervades the chromatin compartment both between and within CTs. Small-scale chromatin loops of the perichromatin region can protrude into the interior of IC channels allowing direct contacts between CDs in cis and trans. At other sites the IC expands to wider, chromatin-free lacunas with splicing speckles and nuclear bodies. This model is in line with a fractal higher-order chromatin arrangement at all levels from CTs, chromosome arms and bands to ~1 Mbp CDs organized as fractal globules (Mirny 2011 Chromosome Res. 19, 37–51). This work is supported by the DFG (ZA 425/1-3, CR 59/29-2).

A high proportion of bovine oocytes fertilized in vitro fail to develop beyond the first 4 cleavage cycles. The first mitotic division of the zygote and proper segregation of chromosomes and cytoplasmic components seems to be a particularly delicate task. Notably, zygotes cleaving with a delay of only a few hours seem to have a very low chance of developing to the blastocyst stage. But what exactly goes wrong, how often, and why? To answer such questions we have to visualize in greater detail basic structures and processes such as the sperm aster, DNA replication, migration and apposition of the 2 pronuclei, synchronous chromosome condensation and breakdown of the nuclear envelopes, assembly of the first mitotic spindle and chromosome congression, anaphase, and cytokinesis. Oocytes fertilized in vitro were fixed at different time points around the first cleavage and stained for DNA, Ser10-phosphorylated histone H3, microtubules, and microfilaments. Zygotes were imaged in toto by recording confocal serial sections at 1-µm intervals using a 40× objective (NA = 1.3). Details were recorded with high spatial sampling densities (pixel size 50 × 50 nm, z-step size of 200 nm) close to the Nyquist criterion and restored by maximum likelihood estimation deconvolution using the real point spread function. We present a series of 3-D confocal images captured at different stages of the first cleavage. The images reveal new insights into the formation, structure, and function of the first mitotic spindle and the occurrence of spindle aberrations, irregular chromosome segregation, and abnormal cytokinesis. The microscopic findings guide us to candidate proteins for localization analyses and functional studies based on 3-D fluorescence live-cell imaging of zygotes and early embryos. This work is supported by the Deutsche Forschungsgemeinschaft (DFG FOR 1041).

As part of a study on structural, molecular, and functional deficiencies of both in vitro and in vivo matured oocytes, the outcome of in vitro fertilization and embryo culture was evaluated by noninvasive time-lapse monitoring. Oocytes for IVM were recovered from abattoir-derived ovaries (G1). In vivo matured oocytes were obtained by ovum pick-up from six FSH (Pluset®, Laboratorios Calier, Spain) superstimulated Simmental heifers (Reichenbach et al. 2010 Reprod Domest Anim. 45, 41) shortly before ovulation (G2). Gonadotropin-releasing hormone (GnRH; Receptal®, MSD, Germany) was applied 18 h before ovum pickup (OPU). After IVM (G1) for 23 h or directly after OPU (G2), all oocytes were fertilized and cultured in vitro for 7 days. To continuously monitor multiple individual embryos in parallel, a well-of-wells culture dish system and a transmitted-light microscope equipped with a digital camera placed in the incubator (Primo Vision, Cryo Innovation Inc., Budapest, Hungary) was used. Each embryo was photographed every 5 min over the whole culture period to precisely determine the time and morphological pattern of the first cleavage and to observe the development up to the hatching blastocyst stage. In a first set of experiments, 128 (G1) and 64 (G2) embryos were cultured and monitored under identical conditions. In a statistical analysis (ANOVA, SAS, SAS Institute Inc., Cary, NC, USA) only embryos with a morphologically regular first and second cleavage (G1: n = 86; G2; n = 42; Total n = 128) were included. Fixed effects in the model were time of first cleavage (class 1: ≤26 hours post-insemination (hpi), class 2: >26 to 28 hpi, class 3: >28 to 30 hpi, class 4: >30 to 32 hpi, class 5: >32 hpi) and in vitro versus ex vivo maturation on blastocyst outcome. The total blastocyst rate was lower after in vitro maturation (40/86) than after in vivo maturation (29/42), while the time of first cleavage was not different. The time until the onset of the first cytokinesis ranged in both oocyte groups from 25–37 hpi. Notably, zygotes that cleaved between 26 and 28 hpi (class 2) showed the highest blastocyst rate (35/47), while the rate decreased in a statistically significant way (P

Establishment of pregnancy in mammals requires prolongation of luteal life span for sustained progesterone (P4) production. P4 stimulates secretory functions of the endometrium required for conceptus growth and development and is essential for endometrial receptivity. The porcine conceptus undergoes rapid differentiation and expansion of its trophoblastic membranes between Day 11 and Day 12 of gestation. With trophoblast elongation production of estrogen, the porcine embryonic pregnancy recognition signal, by the conceptus increases. A complex interplay of estrogen signaling and prostaglandin (PG) metabolism in the endometrium finally results in prevention of luteolysis. Conceptus attachment to the uterine surface epithelium starts around Day 14 of pregnancy, preceded by a pronounced vascularization at the implantation zones, initiating the epitheliochorial placentation. To characterize the complex transcriptome changes in the endometrium in the course of maternal recognition of pregnancy and initial placentation, deep sequencing of endometrial RNA samples of pregnant animals (n = 4) and corresponding non-pregnant controls (n = 4) was performed on Day 12 and Day 14 after ovulation using Illumina RNA-Seq (Illumina Inc., San Diego, CA, USA). Between 21 and 36 million 76-bp single-end reads were produced per sample. Reads were mapped with TopHat to the porcine genome assembly Sscrofa10.2 and relative expression values were calculated based on the National Center for Biotechnology Information’s gene annotation for the analysis of differential gene expression. Statistical analysis revealed more than 3500 differentially expressed genes (DEG) for Day 12 and almost 2500 DEG for Day 14 of pregnancy (DESeq, FDR 1%). Differential expression of selected genes was validated by the use of quantitative real-time PCR (RT-PCR). Comparison of the RNA-Seq data from Day 12 and Day 14 of pregnancy based on a comparison of the DEGs and of overrepresented functional categories revealed distinct differences in gene regulation, reflecting the different functions of the endometrium during these stages; that is, recognition of pregnancy on Day 12 and preparation for conceptus implantation on Day 14. The data from Day 14 was also compared to the published microarray data set from Day 14 of pregnancy (Østrup et al. 2010 Biol. Reprod.) and other sets of DEG by the use of gene set enrichment analysis (GSEA). Overall, a very good agreement with the results of the previous microarray study was found. Furthermore, a significant overlap with genes upregulated in bovine endometrium on Day 18 of pregnancy was found for the genes upregulated in porcine endometrium on Day 14. More than half of these genes were known interferon-regulated genes. In conclusion, RNA-Seq revealed distinct transcriptome changes on Day 12 and Day 14 of pregnancy in porcine endometrium associated with MRP and initiation of implantation.

Interferon-τ (IFNT), a Type-I interferon (IFN), is the pregnancy recognition signal produced by the ruminant conceptus (Godkin et al. 1984; Hansen et al. 1985; Helmer et al. 1987; Spencer et al. 2007). In addition to these specific functions of IFNT in ruminants, many studies suggest that IFNs play a general role in establishment of pregnancy and conceptus attachment/implantation in most mammalian species (Bazer et al. 2009; Bazer et al. 2010; Johnson et al. 2009; Roberts et al. 2008). To characterise the effects of prototype Type-I IFNs on bovine endometrium, in experiment one, Simmental heifers were treated from Day 14 to Day 16 of the oestrous cycle with a rod-shaped intrauterine device releasing human interferon-α (IFNA) or placebo lipid extrudates or PBS only as controls (n = 4 each). Lipid formulation and concentration of human IFNA were adjusted to release 8–9 × 107 IU of IFNA over a period of 2 days in in vitro release experiments. On Day 16, endometrial biopsy samples were collected after flushing the uterus. In experiment 2, endometrial tissue samples were obtained on Day 12, 15 and 18 post-mating from nonpregnant or pregnant heifers. All samples from both experiments were analysed with an Affymetrix Bovine Genome Array (Santa Clara, CA). In experiment one, IFNA treatment resulted in differential gene expression in the bovine endometrium. Significant differences were found between the IFNA group and both control groups, whereas no differences were observed between the placebo and the PBS control group. In experiment 2, differentially expressed genes were found between pregnant and nonpregnant endometria on Day 15 and 18, but not on Day 12, with many of them known IFN-stimulated genes. The comparison of the data sets from both experiments showed very similar gene expression changes for most of the typical IFN-stimulated genes. In addition, several genes were identified which were differentially expressed after IFNA treatment but not different at Day 15 or 18 of pregnancy compared with nonpregnant animals. Conversely, some genes were found as differentially expressed during pregnancy but not after IFNA treatment. Differential expression of selected genes was verified by quantitative real-time PCR and 4 genes, namely jumonji C domain containing histone demethylase 1 homologue D (JHDM1D), indoleamine 2,3-dioxygenase 1 (IDO1), fatty acid binding protein 3, muscle and heart (mammary-derived growth inhibitor) (FABP3) and dickkopf homologue 1 (DKK1), were selected for localization of mRNA expression in endometrial tissue sections. The findings of this study suggest that there may be differential effects of bovine IFNT compared with human IFNA and that some pregnancy-specific changes in the endometrium are elicited by conceptus-derived factors other than IFNT. This study was supported by the German Ministry for Education and Research (BMBF, FUGATO-plus, COMPENDIUM) and the German Research Foundation (DFG FOR478). The authors are part of the European Union COST action GEMINI.

Recent findings on oogenesis, folliculogenesis, and ovarian aging in cows make the bovine system an attractive model for elucidating ovarian function and dysfunction as well as reproductive aging in women. The aim of the present study was to investigate the influence of donor age on the developmental competence of in vitro v. in vivo matured bovine cumulus–oocyte complexes (COC) obtained by ultrasound-guided repeated ovum pickup (OPU). Two groups (G1 and G2) of German Simmental heifers (14 months old at the beginning of the experiment, n = 5 and n = 7), first-lactation young cows (2–4 y old, n = 5 and n = 3), and old cows (10–15 y old, n = 5 and n = 3) were subjected to twice-weekly OPU without hormonal prestimulation 32 (G1) and 6 times (G2). Afterward, animals in G1 were punctured at 5-week intervals 9 times after FSH superstimulation to obtain in vivo matured COC at the metaphase II stage. Data were analysed using a mixed model (SAS). In the twice-weekly OPU for G1 and G2 combined, significantly (P

An alternative approach to classic techniques for the generation of transgenic livestock is the use of viral vectors. Using lentiviral vectors (LV) we previously generated transgenic founder cattle with integrants carrying phosphoglycerate kinase (PGK) promoter-enhanced green fluorescent protein (eGFP) expression cassettes (Hofmann et al. 2004 Biol. Reprod. 71, 405-409). The aim of this work was to investigate the transmission of LV-PGK-eGFP integrants through the female and male germ line of transgenic founder cattle in resulting embryos, fetuses, and offspring. The female founder animal was superovulated and artificially inseminated with a nontransgenic bull. Six of the 16 embryos obtained were transferred to synchronized recipient heifers, resulting in 2 pregnancies and birth of 1 healthy male transgenic calf, expressing eGFP as detected by in vivo imaging and real-time PCR. Cryopreserved semen of the founder bull and matured COC of nontransgenic cows were used for in vitro embryo production as previously described by Hiendleder et al. (2004 Biol. Reprod. 71, 217-223). The rates of cleavage and development to blastocysts in vitro corresponded to 52.3 ± 3.8% and 23.5 ± 4.6%, respectively. In vivo expression of eGFP was observed at blastocyst stage (Day 7 after IVF) and was seen in 93.8% (198/211) of all blastocysts. Twenty-four eGFP-positive embryos were transferred to 9 synchronized recipients. Analysis of 2 embryos flushed on Day 15, 2 fetuses recovered on Day 45, and a healthy male transgenic calf revealed consistent high-level expression of eGFP in all tissues investigated. These observations show for the first time transmission of lentiviral integrants through the germ line of female and male transgenic founder cattle. Although eGFP transgenic cattle have been produced before by nuclear transfer from transfected cells, lentiviral transgenesis has the advantage that only one copy of the provirus is integrated at a particular chromosomal integration site. High-fidelity expression of eGFP in embryos, fetuses, and offspring of founders provides an interesting tool for developmental studies in cattle, including interactions of gametes, embryos, and fetuses with their maternal environment.