In nuclear transfer (NT) embryos, exposure of the donor chromatin to the MII cytoplasm results in premature chromatin condensation (PCC) which may be beneficial for nuclear reprogramming (Campbell KHS and Alberio R 2003 Reprod. Suppl. 61, 477–494). Following enucleation, maturation promoting factor (MPF) activity in murine oocytes is primarily associated with the meiotic spindle. This reduced MPF activity in the cytoplast may result in decreased PCC and reprogramming. Conversely, increasing cytoplast MPF activity may increase reprogramming. The aims of this study were to perform quantitative analysis of MPF and MAPK activities in ovine oocytes: 1. at anaphase/telophase I (A/TI) or MII; 2. following enucleation; 3. following treatment with caffeine (an inhibitor of Myt1/Wee1 activity). The development of ovine NT embryos reconstructed using caffeine-treated oocytes as cytoplast recipients was then determined. Oocytes were matured in TCM 199, 10% FBS, 5μgmL−1 FSH, 5μgmL−1 LH,1μgmL−1 estradiol, 0.3mM sodium pyruvate and 100μM cysteamine. 15h post-onset of maturation (hpm) oocytes were stripped of cumulus cells and enucleated in HSOF containing 5μgmL−1 Hoechst 33342 and 7.5μgmL−1 cytochalasin B (CB). Control oocytes were sham-enucleated by removing an equal volume of cytoplasm. Oocytes were cultured in SOF±10mM caffeine. Groups of 10 oocytes were sampled and analyzed for MPF and MAPK activities as previously described (Ye JP et al., 2003 Reproduction 125, 645–656). For NT, primary foetal fibroblasts were quiesced in DMEM containing 0.1% FBS for 2–3 days. Cell fusion was induced with two DC pulses of 25VcM-1 for 80μs. 3 methods of NT were compared: A. fusion 20hpm, activation 21hpm; B. fusion 24hpm, activation 25hpm; C. 10mM caffeine 18–24hpm, fusion 24hpm, activation 25hpm. All oocytes were activated in HSOF containing 5μgmL−1 calcium ionophore (A23187), cultured in SOF with 10μgmL−1 of cycloheximide and 7.5μgmL−1 CB for 5h, and then transferred to mSOFaaBSA medium, all at 5% CO2, 5% O2 and 90% N2 at 39°C. On Day 2 cleavage was assessed and 10% FBS added to the medium. Development to blastocyst was assessed on Day 7. All data were analyzed by chi-square test. Both MPF and MAP kinase activities were increased at MII compared to A/TI (P<0.05). There were no differences in activities of both kinases between intact and enucleated oocytes. Following enucleation, both kinase activities were identical in all groups, reaching maximum activities 24hpm followed by a slow decline. Caffeine increased the activity of both kinases (MPF in particular) in all groups. Following 5 replicates (total oocytes 145, 143, 144 for NT methods A,B,C, respectively), no significant differences were observed between fusion (82.1%, 67.8%, 67.4%), cleavage (90.8%, 88.7%, 89.7%) or development to blastocyst (20.2%, 18.6%, 25.8%). Analysis of total cell numbers on limited numbers of blastocysts (7, 6, 7) were NS(70.9±38.5, 69.3±25.4, 93.3±17.8).

In nuclear transfer reconstructed embryos, the coordination of donor nuclear and recipient cytoplasmic cell cycle phases is essential to maintain ploidy and prevent DNA damage. However, the stage of the cell cycle at the time of reconstruction and the method of reconstruction may also have a significant impact on the subsequent development of the embryo and fetus through a number of other mechanisms. This paper reviews some of the information currently available and proposes that consideration of the cell cycle may lead to improvement of methods for embryo reconstruction.

The superstimulation protocol of Blondin et al. (2002; Biol. Reprod. 66, 38-43) produces cumulus-oocyte complexes (COCs) of high developmental competence for IVP. Using a similar protocol we assessed the affects of alterations in oocyte donor carbohydrate and lipid metabolism during ovarian stimulation on the production and viability of blastocysts in vitro. A 2 × 2 factorial experiment offered two diets: Fiber (F) and Starch (S) alone (0) or with 6% w/w (6) protected lipid (calcium soaps of fatty acids). Thirty-two heifers ranked by body condition score (scale: 1 = thin, 5 = obese) were allocated within score to one of the 4 treatments: F0, F6, S0 and S6. COCs were collected 5 days after estrus by OPU for lipid analysis. Ovarian stimulation (4 doses of FSH (9 mg NIADDK oFSH) given 12 h apart) commenced 2 days later. COCs were collected 40 h after the last FSH injection. GnRH (0.012 mg Buserelin) was administered i.v. 6 h prior to OPU. A second period of ovarian stimulation and OPU then followed. Following IVM/IVF, zygotes were cultured in SOF with 0.3% w/v fatty acid-free BSA under oil (38.8 • C, 5% CO 2 , 5% O 2 , 90% N) until Day 8 of development, when blastocysts were subjected to total cell counts and TUNEL analysis. Data were analyzed by ANOVA. Neither follicles aspirated (25.9 ± 1.87) nor oocytes recovered (12.1 ± 0.92) differed between treatments. Total fatty acids in plasma were greater (P < 0.001) for the F than for the S diets and increased with the inclusion of protected lipid (0.75, 1.82, 0.50 and 1.39 µg mL −1 for F0, F6, S0 and S6, respectively; SED = 0.076). The dietary lipid-induced increase in plasma fatty acids was reflected in an increase (P < 0.05) in total fatty acids within the oocyte (70.4, 74.7, 69.9 and 78.4 ng/oocyte; SED = 3.41). Retrospective analysis by body condition indicated that S diets reduced (P = 0.006) blastocyst yields in thin heifers and reduced (P = 0.02) cleavage rates in fat heifers (Table 1). Blastocyst yields were lower (P = 0.1) for fat heifers on the F0 diet. Total cell numbers

Currently techniques of TUNEL labelling for detection of apoptosis and differential staining for counting the ratio of inner cell mass (ICM) to trophoectoderm (TE) cells are used separately for assessment of embryo quality in different species. The majority of these techniques are antibody-based, and time-consuming, frequently giving inconsistent results. Here we report on the development of a simple and fast method for simultaneous differential staining and TUNEL labelling of bovine embryos. Cleaved embryos produced from in vitro-matured and fertilized oocytes were cultured to the blastocyst stage in synthetic oviductal fluid culture medium (SOF) supplemented with 4mgmL−1 BSA and 5% FCS. Embryos were partially permeabilized in 0.5% Triton X-100 solution containing 2μMmL−1 TOTO-3 dye (Molecular Probes, Eugene, OR, USA) for 30s. TOTO-3 is a cell-impermeant nucleic acid dye; thus only permeabilized cells are stained red. The embryos were then quickly washed in PBS containing 3mgmL−1 PVA, fixed for 15min at RT in 4% paraformaldehyde containing 10μgmL−1 Hoescht, and TUNEL-labelled using a Cell Death Kit (Roche Applied Science, East Sussex, UI) for 30min at 37°C in a humid chamber. The embryos were then treated with RNase A (50UmL−1) for 30min at 37°C, washed and mounted in a small drop of glycerol on a glass slide. RQ1-DNase (3UmL−1)-treated embryos were used as a positive control. After three-dimensional reconstruction using a Leica TCS SP2 confocal microscope, we determined the number of ICM (blue), TE (red) and apoptotic nuclei (green). Only peripheral cells of the blastocysts were labelled red, indicating that TE cells were permeabilized by the short exposure to the detergent Triton. ICM cells were consistently stained blue by the cell permeant dye Hoechst. Apoptotic nuclei were found in both types of cells. More consistent differential staining was observed in hatched blastocysts (n=30) than in zona-enclosed blastocysts (n=35); also, more apoptotic nuclei were observed. No differences were found in the consistency of the technique for embryos grown with or without FCS. When compared to dual staining without Tunel, no differences in cell number (74±22) , and ICM/TE ratio (0.28±0.06) were detected, indicating that the Tunel procedure does not affect the labeling of the DNA. Preliminary observations also indicate that this method can be successfully applied to porcine and ovine embryos. This technique has the advantage of being fast and can be applied for assessment of embryo quality. It can also be used to determine the time and origin of ICM and TE differentiation while monitoring the degree of apoptosis in different culture systems and in different species. This work was in part supported by Department of Environment Food and Rural Affairs (Defra) UK.

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