Phosphofructokinase and Malate Dehydrogenase Participate in the In Vitro Maturation of Porcine Oocytes

Oocyte maturation depends on the metabolic activity of cumulus–oocyte complex (COC) that performs nutritive and regulatory functions during this process. In this work, the enzymes [phosphofructokinase (PFK) and malate dehydrogenase (MDH)] were tested to elucidate the metabolic profile of porcine COCs during the in vitro maturation (IVM). Enzymatic activity was expressed in U/COC and U/mg protein (specific activity) as mean ± SEM. In vitro maturation was performed with 2‐oxoglutarate (5, 10 and 20 mm ) or hydroxymalonate (30, 60 and 100 mm ) inhibitors of PFK and MDH, respectively. The PFK and MDH activities (U) remained constant during maturation. For PFK, the U were (2.48 ± 0.23) 10^?5 and (2.54 ± 0.32) 10^?5, and for MDH, the U were (4.72 ± 0.42) 10^?5 and (4.38 ± 0.25) 10^?5 for immature and in vitro matured COCs, respectively. The specific activities were significantly lower after IVM, for PFK (4.29 ± 0.48) 10^?3 and (0.94 ± 0.12) 10^?3, and for MDH (9.08 ± 0.93) 10^?3 and (1.89 ± 0.10) 10^?3 for immature and in vitro matured COCs, respectively. In vitro maturation percentages and enzymatic activity diminished with 20 mm 2‐oxoglutarate or 60 mm hydroxymalonate (p < 0.05). Viability was not affected by any concentration of the inhibitors evaluated. The U remained unchanged during IVM; however, the increase in the total protein content per COC provoked a decrease in the specific activity of both enzymes. Phosphofructokinase and MDH necessary for oocyte IVM would be already present in the immature oocyte. The presence of inhibitors of these enzymes impairs the meiotic maturation. Therefore, the participation of these enzymes in the energy metabolism of the porcine oocyte during IVM is confirmed in this study.     

N2O emissions from different cropping systems and from aerated,nitrifying and denitrifying tanks of a municipal waste water treatment plant

Nitrous oxide emissions, nitrate, water-soluble carbon and biological O₂ demand (BOD₅) were quantified in different cropping systems fertilized with varying amounts of nitrogen (clayey loam, October 1991 to May 1992), in an aerated tank (March 1993 to March 1994), and in the nitrification-denitrification unit (March to July 1994) of a municipal waste water treatment plant. In addition, the N₂O present in the soil body at different depths was determined (February to July 1994). N₂O was emitted by all cropping systems (mean releases 0.13–0.35 mg N₂O m^-2 h^-1), and all the units of the domestic waste water treatment plant (aerated tank 0–6.2 mg N₂O m^-2 h^-1, nitrification tank 0–204,3 mg N₂O m^-2, h^-1, denitrifying unit 0–2.2 mg N₂O m^-2 h^-1). During the N₂O-sampling periods estimated amounts of 0.9, 1.5, 2.4 and 1.4 kg N₂O–N ha^-1, respectively, were released by the cropping systems. The aerated, nitrifying and denitrifying tanks of the municipal waste water treatment plant released mean amounts of 9.1, 71.6 and 1.8 g N₂O–N m^-2, respectively, during the sampling periods.The N₂O emission were significantly positively correlated with nitrate concentrations in the field plots which received no N fertilizer and with the nitrogen content of the aerated sludge tank that received almost exclusively N in the form of NH ₄ ⁺ . Available carbon, in contrast, was significantly negatively correlated with the N₂O emitted in the soil fertilized with 80 kg N ha^-1 year. The significant negative correlation between the emitted N₂O and the carbon to nitrate ratio indicates that the lower the carbon to nitrate ratio the higher the amount of N₂O released. Increasing N₂O emissions seem to occur at electron donorto-acceptor ratios (C_H2_O or BOD₅-to-nitrate ratios) below 50 in the cropping systems and below 1200–1400 in the waste water treatment plant. The trapped N₂O in the soil body down to a depth of 90 cm demonstrates that agricultural production systems seem to contain a considerable pool of N₂O which may be reduced to N₂ on its way to the atmosphere, which may be transported to other environments or which may be released at sometime in the future.Dedicated to Professor J.C.G. Ottow on the occasion of his 60th birthday     

Use of ultrasonography to diagnose Sertoli cell neoplasia and cryptorchidism in a dog

Unilateral cryptorchidism and Sertoli cell tumor in the descended testis were provisionally diagnosed by use of ultrasonography in a 7-year-old Keeshond. The left testis was thought to have degenerated, but ultrasonography of the scrotum did not reveal evidence of testicular or epididymal tissue. The contralateral testis contained a hypoechoic mass believed to be a testicular tumor. Surgery and histopathologic findings confirmed the diagnosis. Ultrasonography of the scrotum and its contents should be considered for use in animals with suspected testicular neoplasia and/or degeneration.     

Infectivity and virulence of Australian strains of Moraxella bovis for the murine and bovine eye in relation to pilus serogroup sub-unit size and degree of piliation

The degree of piliation of 29 haemolytic and 4 non-haemolytic Australian strains of Moraxella bovis representing 7 different pilus antigen groups was determined. The infectivity and virulence for the eye was measured in steroid-treated mice and in cattle. Non-piliated strains failed to infect the murine eye. Most moderately or heavily piliated strains reproducibly produced the highest infectivity and virulence scores in mice when compared with lightly or very lightly piliated strains (p less than 0.05). Non-haemolytic, piliated strains were infective and in one instance virulent for mice. Almost similar levels of infectivity and virulence were observed for 7 representative haemolytic strains tested in both cattle and mice. The relative molecular weight of pilin sub-units was compared using sodium dodecyl-sulphate polyacrylamide gel electrophoresis. Three classes of pili, alpha, beta and gamma of ascending sub-unit size were identified among the 7 pilus antigen serogroups. Pilin sub-unit size bore no relationship to the degree of piliation but most strains that were highly virulent in mice and cattle expressed alpha and gamma sub-units. Some strains appeared capable of switching from alpha to beta or form beta to gamma sub-unit production.     

Heat Shock Protein 70 and Sex Steroid Receptors in the Follicular Structures of Induced Ovarian Cysts

The purpose of this study was to estimate the expression and relative amounts of estrogen (ER) and progesterone receptors (PR) and their isoforms as well as heat shock protein 70 (HSP70) in ovaries of rats with induced cystic ovarian disease (COD). Primary, secondary, tertiary, atretic and cystic follicles were evaluated by immunohistochemistry and total ovarian proteins were analyzed by Western blot. In the granulosa layer, growing and cystic follicles in the treated group have a higher expression of ERα than growing follicles of control individuals. In the theca interna layer, tertiary follicles presented a significantly higher expression of ERα in the treated group. An increase in total ERα protein was detected in the treated group. Granulosa cells of all growing, atretic and cystic follicles show a lower expression of ERβ in animals with COD, and the total protein expression of ERβ was lower in this group. The expression of PR was lower in the granulosa cell layer of tertiary and cystic follicles in treated animals, and theca interna layer had less intense immunostaining in this group. Although there were no differences in the expression of PR-B by Western blotting, the expression of PR-A was higher and the expression of PR-C was smaller in the treated group. An intense HSP70 immunostaining was observed in the cells of cystic follicles. By Western blotting, higher protein expression of HSP70 was detected in the ovarian samples of the control group than those of the treated ones. Ovaries of animals with COD exhibited an altered steroid receptor expression and subtype balance as compared with control animals, and an increase in HSP70 immunoexpression.     

Persistent breeding-induced endometritis after hysteroscopic insemination in the mare

Low-dose insemination has been proposed to reduce persistent breeding-induced endometritis (PBIE) in mares with delayed uterine clearance (DUC). Others proposed that hysteroscopic insemination induces an exaggerated inflammatory response and should be avoided in DUC mares. The objectives here were to evaluate presence and severity of PBIE in normal and DUC mares after hysteroscopic insemination with fresh semen, and to determine if hysteroscopy could be used in DUC mares without inducing excessive inflammation. Reproductively normal (n = 4) and DUC (n = 5) mares received four treatments in random order: uterine body insemination (UB, 1 × 10(9) spermatozoa, 20 ml), hysteroscopic insemination (HYST, 5 × 10(6) spermatozoa, 0.5 ml), sham hysteroscopic insemination (SHAM, semen extender, 0.5 ml) and hysteroscopic infusion of seminal plasma (SP, 0.5 ml). Significantly more DUC (50%) mares than normal (14%) mares accumulated intrauterine fluid 24 h post-treatment. The difference in fluid accumulation between DUC (40%) mares and normal (7%) mares was also significant 48 h post-treatment. Fluid scores were not significantly different between treatments in normal mares. However, treatments HYST and SHAM resulted in significantly higher fluid scores 24 h but not 48 h post-treatment in DUC mares. There was no effect of treatment or mare group on the percentage and total number of neutrophils in uterine fluid 48 h post-treatment. Percentage of neutrophils was correlated with duration of hysteroscopy in normal mares, with procedures lasting ≥ 9 min associated with PBIE. There was no effect of mare group, treatment or duration of hysteroscopy on pregnancy rate. Hysteroscopy induces a transient inflammation that is not more severe than that after conventional artificial insemination, suggesting no contraindication to its use in DUC mares.     

Testing Usability of Butylated Hydroxytoluene in Conservation of Goat Semen

The objective of this study was to investigate whether butylated hydroxytoluene (BHT) could be used as a suitable supporter or alternative of egg yolk during preservation of goat spermatozoa. Three in vitro experiments and a fertility test were conducted to evaluate the effect of BHT on viability of chilled‐stored semen as well as motility and kidding rate of frozen‐thawed spermatozoa. In the first two experiments, ejaculates (n = 30/experiment) were collected from 10 bucks, split, diluted with egg yolk‐based and egg yolk‐free extenders supplemented with or without 0.3, 0.6, 2, 5 and 8 mm BHT and stored at 5°C for 168 h. In the third experiment, 30 ejaculates were collected from the above‐mentioned bucks, split and diluted with egg yolk‐free extenders supplemented with or without 0.3, 0.6 and 0.9 mm BHT and egg yolk‐based extenders supplemented with or without 5 mm BHT. Diluted semen was cooled to 5°C over a period of 4 h, frozen and thawed in the form of 0.3‐ml pellets. In the fertility test, 75 ejaculates were collected from two proven fertile bucks, split, diluted with egg yolk‐free extenders containing 0.6 mm BHT and egg yolk‐based extenders supplemented with or without 5 mm BHT, frozen and thawed as described above. An insemination volume of 0.6 ml containing 120–140 × 10⁶ progressively motile spermatozoa was used for a single cervical insemination of cloprostenol‐synchronized does (n = 230). The results showed that addition of 5 mm BHT to egg yolk‐deficient (2.5%) extenders significantly improved viability of chilled‐stored semen together with motility (48.5%) and fertility (62.5%) of frozen‐thawed spermatozoa. Replacement of egg yolk in semen extenders by 0.6 mm BHT could sustain not only viability of chilled‐stored semen but also post‐thaw motility (47.5%) and fertility (53.75%) of frozen‐thawed spermatozoa. In conclusion, supplementation of semen diluents with BHT can ameliorate preservability of goat sperm.     

Characterization and cooled storage of semen from corn snakes (Elaphe guttata).

The phylogenetic order Squamata has many representatives that could benefit from the use of semen preservation as a tool for assisting conservation. To date, few studies have been made evaluating the potential for collecting and preserving semen from snakes. The objectives of this study were to characterize semen parameters of the corn snake (Elaphe guttata), including appearance, volume, concentration, sperm motility, and sperm morphology, and to determine the longevity of corn snake sperm motility stored at 4 degrees C. Single semen samples were collected from 22 adult corn snakes. The appearance of the corn snake semen was generally cloudy, and the color was white to tan. Corn snake spermatozoa initially exhibited a median motility of 92.5%. Corn snakes were found to produce small-volume ejaculates (median 0.01 ml). However, the overall concentration of the snake ejaculate was high (chi = 852 x 10(6) +/- 585 x 10(6) spermatozoa/ml). Morphologically, a mean of 75.7 +/- 9.3% of the sperm cells in an ejaculate were normal. Snake ejaculate with a white appearance had significantly higher sperm concentrations (chi = 1,859 x 10(6) +/- 1,008 x 106 sperm cells/ml; F = 15.74, P = 0.001) than tan ejaculates (chi = 601 x 10(6) +/- 439 x 106 sperm cells/ml). Sperm motility decreased significantly in samples that were stored at 4 degrees C for greater than 48 hr in a refrigerator or Equitainer I. This is the first study to characterize semen volume, appearance, and concentration; sperm motility; and sperm morphology in captive corn snakes. The information derived from this study can be used to develop a model for a collection, cooled storage, and shipping program for semen from endangered or threatened captive and wild snakes.     

A "Schrodinger Cat" Superposition State of an Atom

A "Schrodinger cat"-like state of matter was generated at the single atom level. A trapped 9Be+ ion was laser-cooled to the zero-point energy and then prepared in a superposition of spatially separated coherent harmonic oscillator states. This state was created by application of a sequence of laser pulses, which entangles internal (electronic) and external (motional) states of the ion. The Schrodinger cat superposition was verified by detection of the quantum mechanical interference between the localized wave packets. This mesoscopic system may provide insight into the fuzzy boundary between the classical and quantum worlds by allowing controlled studies of quantum measurement and quantum decoherence.