Fertilization Ability of Porcine Oocytes Reconstructed from Ooplasmic Fragments Produced and Characterized after Serial Centrifugations

Mitochondria are reported to be critical in in vitro maturation of oocytes and subsequent embryo development after fertilization, but their contribution for fertilization has not been investigated in detail. In the present study, we investigate the contribution of mitochondria to fertilization using reconstructed porcine oocytes by fusion of ooplasmic fragments produced by serial centrifugations (centri-fusion). Firstly, we evaluated the characteristics of ooplasmic fragments. Three types of fragments were obtained by centrifugation of porcine oocytes matured in vitro for 46 h: brownish (B), transparent (T) and large (L) fragments containing both B and T parts in a fragment. The production efficiencies of these types of fragments were 71.7, 91.0 and 17.8 fragments/100 oocytes, respectively. In experiments, L fragments were excluded because they contained both brownish and transparent components that were apparently intermediate between B and T fragments. Observations by confocal microscopy after staining with MitoTracker Red CMXRos^® and transmission electron microscopy revealed highly condensed active mitochondria in B fragments in contrast to T fragments that contained only sparse organelles. We reconstructed oocytes by fusion of a karyoplast and two cytoplasts from B and T fragments (B and T oocytes, respectively). The B oocytes showed higher sperm penetration (95.8%) and male pronuclear formation rates (94.2%) by in vitro fertilization than T oocytes (66.7% and 50.0%, respectively). These results suggest that the active mitochondria in oocytes may be related to their ability for fertilization.     

Influence of fentanyl on intra-abdominal pressure during laparoscopy in dogs

ObjectiveTo evaluate the influence of fentanyl on intra-abdominal pressures in spontaneously breathing dogs during capnoperitoneum.Study designProspective clinical study.AnimalsEleven healthy client-owned and five healthy experimental dogs undergoing laparoscopy.MethodsDogs were premedicated with acepromazine (0.03 mg kg^?1 IV) and carprofen (4 mg kg^?1 IV). Anaesthesia was induced with propofol and maintained with isoflurane in oxygen. The abdomen was insufflated with CO₂ (11–16 cm H₂O). Intra-abdominal pressures were measured with a transducer. Respiratory variables were measured with a spirometry sensor and side-stream capnography. Following preparation, fentanyl (1 μg kg^?1) was injected over 30 seconds IV. Data were recorded 5 minutes before, during and 5 minutes after treatment. The following time points were selected for statistical analysis (anova, p < 0.05): ?160, ?140, ?120, ?100, ?80, ?60, ?40, ?20, 0, 30, 50, 70, 90, 110, 130 and 150 seconds after the start of fentanyl injection.ResultsIntra-abdominal pressure increased during inspiration in 15 dogs but decreased in one dog. Fentanyl treatment did not alter these patterns. Peak inspiratory and end-expiratory intra-abdominal pressures continuously decreased over time during the whole experiment and fentanyl exaggerated the decrease in inspiratory pressures but did not affect the rate of decrease in expiratory pressures. Differences between intra-abdominal pressures were stable before, but decreased after fentanyl administration from 4.1 ± 1.4 to 3.3 ± 1.2 cm H₂O (at 0 and 150 seconds time points). End-tidal CO₂ partial pressures increased from 6.0 ± 0.8 to 6.6 ± 0.9 kPa, respiratory rate decreased from 10.8 ± 2.6 to 7.8 ± 2.2 breaths per minute and tidal volume decreased from 13.7 ± 4.4 to 12.4 ± 2.9 mL kg^?1 after fentanyl but these variables did not change before fentanyl treatment. Airway pressures did not change.Conclusions and clinical relevanceFentanyl did not increase intra-abdominal pressures in dogs.     

Maturation ability after transfer of freeze‐dried germinal vesicles from porcine oocytes

The purpose of this study was to examine whether freeze‐dried germinal vesicles (GV) can be matured in vitro after being injected into enucleated fresh oocytes in pigs as an alternative method for conservation of genetic resources. Although no reduction of the size of GV (p = .094), resveratrol treatment significantly enhanced the survival rates following GV transfer (GVT) (p < .001). Supplementation with 100 or 200 mmol/L trehalose in freeze‐drying medium significantly increased the proportions of GVs with intact nuclear membrane and DNA integrity compared with the control group. Following transfer of freeze‐dried GVs into enucleated fresh oocytes, the proportion of reconstructed oocytes reached the metaphase‐II stage (2.4% ± 1.4%) was significantly lower (p < .05) than that of the in vitro matured control group (83.2% ± 2.5%), it was comparable with the GVT control group (7.4% ± 2.7%). The rates of freeze‐dried GVs with intact nuclear membrane and DNA stored at ?20°C for 5 days were significantly higher (p < .05) than those at 4°C and room temperature. The rates of intact nuclear membrane and DNA in the freeze‐dried GV stored for 15 or 30 days at ?20, 4°C and RT were not significantly different. In conclusion, matured oocytes were produced derived from freeze‐dried GVs.     

Milrinone treatment of bovine oocytes during in vitro maturation benefits production of nuclear transfer embryos by improving enucleation rate and developmental competence

In the production of cattle nuclear transfer embryos, the production efficiency is affected by the oocyte developmental competence and successful enucleation rate. This study investigated the effect of treating oocytes with milrinone, a phosphodiesterase inhibitor, on these two characteristics. When cumulus-oocyte complexes (COCs) were cultured for 19 h with 0, 50 or 100 μM of milrinone, the enucleation rate was significantly improved by 100 μM milrinone. However, milrinone treatment during in vitro maturation (IVM) also delayed meiotic progression by at least 2 h, which would affect the examination of enucleation rate and developmental competence of oocytes. Thus, in the second experiment, meiotic resumption was temporarily inhibited with butyrolactone I (BL-I; 100 μM, 18 h) to decrease the delayed maturation caused by milrinone; this enabled a more accurate comparison of the effects of milrinone after oocyte maturation. In nuclear transfer embryo production, oocytes treated with milrinone (100 μM, 20 h) showed a significantly higher rate of enucleation compared with that of control oocytes. This improved enucleation rate was associated with a closer location of the metaphase plate to the first polar body in the treated oocytes compared with that in control oocytes. Furthermore, milrinone improved the frequency of development to the blastocyst stage in the resulting embryos. In conclusion, milrinone supplementation during IVM improved enucleation rates by rendering the metaphase plate in close proximity to the first polar body, and this treatment also improved oocyte developmental competence. These benefits additively improved the yield of cloned embryos that developed to the blastocyst stage.     

Cryopreservation of immature buffalo oocytes: Effects of cytochalasin B pretreatment on the efficiency of cryotop and solid surface vitrification methods

The aim of the present study was to compare the efficiency of the solid surface (SSV), cryotop (CT) vitrification methods and cytochalasin B (CB) pretreatment for cryopreservation of immature buffalo oocytes. Cumulus‐oocyte complexes (COCs) were placed for 1 min in TCM199 containing 10% dimethylsulfoxide (DMSO), 10% ethylene glycol (EG), and 20% fetal bovine serum, and then transferred for 30 s to base medium containing 20% DMSO, 20% EG and 0.5 mol/L sucrose. CB pretreated ((+)CB) or non‐pretreated ((?)CB) COCs were vitrified either by SSV or CT. Surviving vitrified COCs were selected for in vitro maturation (IVM) and in vitro fertilization (IVF). The rate of viable oocytes after vitrification in CT groups (82%) was significantly lower (P < 0.05) than that in a fresh control group (100%), but significantly higher (P < 0.05) than those in SSV groups (71–72%). Among vitrified groups, the highest maturation rate was obtained in the CT (?)CB group (32%). After IVF, the cleavage and blastocyst formation rates were similar among vitrified groups but significantly lower than those of the control group. In conclusion, a higher survival rate of oocytes after vitrification and IVM was obtained in the CT group compared with that in the SSV group, indicating the superiority of the CT method. Pretreatment with CB did not increase the viability, maturation or embryo development of vitrified oocytes.     

Rapid rewiring of arcuate nucleus feeding circuits by leptin

The fat-derived hormone leptin regulates energy balance in part by modulating the activity of neuropeptide Y and proopiomelanocortin neurons in the hypothalamic arcuate nucleus. To study the intrinsic activity of these neurons and their responses to leptin, we generated mice that express distinct green fluorescent proteins in these two neuronal types. Leptin-deficient (ob/ob) mice differed from wild-type mice in the numbers of excitatory and inhibitory synapses and postsynaptic currents onto neuropeptide Y and proopiomelanocortin neurons. When leptin was delivered systemically to ob/ob mice, the synaptic density rapidly normalized, an effect detectable within 6 hours, several hours before leptin's effect on food intake. These data suggest that leptin-mediated plasticity in the ob/ob hypothalamus may underlie some of the hormone's behavioral effects.     

Saturn's variable magnetosphere

Since the Cassini spacecraft reached Saturn's orbit in 2004, its instruments have been sending back a wealth of data on the planet's magnetosphere (the region dominated by the magnetic field of the planet). In this Viewpoint, we discuss some of these results, which are reported in a collection of reports in this issue. The magnetosphere is shown to be highly variable and influenced by the planet's rotation, sources of plasma within the planetary system, and the solar wind. New insights are also gained into the chemical composition of the magnetosphere, with surprising results. These early results from Cassini's first orbit around Saturn bode well for the future as the spacecraft continues to orbit the planet.     

Stickstoffverlagerung unter spätbeweidetem Grünland

Leaching of nitrogen from pastures at the end of the grazing season A trial was carried out to describe nitrogen dynamics under excrement patches. On three grassland sites differing in water capacity, soil water was extracted by porous ceramic cups placed under the patches. Soil water was analyzed for different nitrogen fractions. Infiltration water and the amount of leached nitrogen was calculated by a simulation model. The rapid rise in concentrations under the urine patches to 30–60 mg NH₄?N/I was due to the rapid hydrolysis of urea in spite of low soil temperatures. While the rates of ammonium decreased, the concentration of nitrate increased continuously up to 160 mg NO₃?N/I and did not fall until the beginning of plant growth in early spring. Under the dung patches almost no nitrogen was found. For the urine patches the calculated nitrogen leaching was between 150 and 320 kg/ha, for the dung patches between 3 and 28 kg/ha. From the total of leached nitrogen the nitrate fraction (83%) was the most significant, followed by the organic nitrogen fraction (11%) and ammonium (6%). Taking account of an estimated grazing pressure, the urine-affected soil surface was calculated between 1% and 3