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.     

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.     

Temperature modulates liver lipid accumulation in Atlantic salmon (Salmo salar L.) fed low dietary levels of long‐chain n‐3 fatty acids

Atlantic salmon (Salmo salar) were fed five graded levels of eicosapentaenoic acid (EPA, 20:5n‐3) and docosahexaenoic acid (DHA, 22:6n‐3), from 1.4 to 5.2% of total fatty acids (FA, 5–17 mg kg^?1 feed), and grew from ~160 g to ~3000 g, with the period from 1450 g onwards conducted both at 6 °C and at 12 °C. All fish appeared healthy, and there were no diet‐related differences in haematological or plasma parameters, as well as intestinal histological or gut microbiota analysis. Fish reared at 6 °C had higher accumulation of storage lipids in the liver compared to fish reared at 12 °C. Liver lipids also increased with decreasing dietary EPA + DHA at 6 °C, while there was no such relationship at 12 °C. Gene expression of SREBP1 and 2, LXR, FAS and CPT1 could not explain the differences in liver lipid accumulation. In liver polar lipids, DHA was found to be reduced when dietary EPA + DHA was <2.7% of FAs, while the level of EPA in the membranes was not affected. In conclusion, reducing dietary EPA + DHA from 5.2 to 1.4% of total FAs had a minor impact on fish health. Temperature was the factor that most affected the liver lipid accumulation, but there was also an interaction with dietary components.     

Dietary fatty acids and inflammation in the vertebral column of Atlantic salmon, Salmo salar L., smolts: a possible link to spinal deformities

Vegetable oils (Vo) are an alternative to fish oil (Fo) in aquaculture feeds. This study aimed to evaluate the effect of dietary soybean oil (Vo diet), rich in linoleic acid, and of dietary fish oil (Fo diet) on the development of spinal deformities under bacterial lipopolysaccharide (LPS)‐induced chronic inflammation conditions in Atlantic salmon, Salmo salar L. Fish [25 g body weight (BW)] were fed the experimental diets for 99 days. On day 47 of feeding (40 g BW), fish were subjected to four experimental regimes: (i) intramuscular injections with LPS, (ii) sham‐injected phosphate‐buffered saline (PBS), (iii) intraperitoneally injected commercial oil adjuvant vaccine, or (iv) no treatment. The fish continued under a common feeding regime in sea water for 165 more days. Body weight was temporarily higher in the Vo group than in the Fo group prior to immunization and was also affected by the type of immunization. At the end of the trial, no differences were seen between the dietary groups. The overall prevalence of spinal deformities was approximately 14% at the end of the experiment. The Vo diet affected vertebral shape but did not induce spinal deformities. In groups injected with LPS and PBS, spinal deformities ranged between 21% and 38%, diet independent. Deformed vertebrae were located at or in proximity to the injection point. Assessment of inflammatory markers revealed high levels of plasma prostaglandin E₂ (PGE₂) in the Vo‐fed and LPS‐injected groups, suggesting an inflammatory response to LPS. Cyclooxigenase 2 (COX‐2) mRNA expression in bone was higher in fish fed Fo compared to Vo‐fed fish. Gene expression of immunoglobulin M (IgM) was up‐regulated in bone of all LPS‐injected groups irrespective of dietary oil. In conclusion, the study suggests that Vo is not a risk factor for the development of inflammation‐related spinal deformities. At the same time, we found evidence that localized injection‐related processes could trigger the development of vertebral body malformations.     

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.     

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.     

Including processed poultry and porcine by‐products in diets high in plant ingredients reduced liver TAG in Atlantic salmon,Salmo salar L.

The effects on Atlantic salmon (Salmo salar L.) metabolic health of including modern processed land animal by‐products (LAP) to a plant‐ and marine‐based diet (50% marine and 50% plant ingredients) were investigated. Three experimental diets were made with systematic replacements of both marine and plant ingredients with LAP as a source of protein (poultry meal and porcine blood meal) and fat (poultry oil) to fit a two‐way factorial design. A 16‐week feeding trial was performed with postsmolts in seawater (initial weight 372 g). The diet with both protein and lipids from LAP reduced liver triacylglycerols more than fourfold compared to the diet without LAP. This was confirmed by histological examinations showing reduced fatty degeneration in the liver of fish fed the high LAP diet. No severe negative effects on gut or tissue health were seen by histological examinations or by measuring genetic markers with qPCR, although a trend in the histological results indicated an improved gut health by including LAP in the diets. Clinical analyses of plasma and lipoprotein fractions showed no differences between dietary groups.     

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.     

Salmon farming affects the fatty acid composition and taste of wild saithe Pollachius virens L.

There is an ongoing controversy on whether fish farming affects the quality of wild fish in fjords. In northern Norway, local people prefer not to eat saithe, Pollachius virens L., from areas in the vicinity of fish farms because they say the taste is inferior to saithe from other areas. To address this issue, saithe were collected in the vicinity of a salmon fish farm in a fjord in northern Norway and in two reference areas: one site 6 km away from the nearest fish farm in the same fjord, and the other in a fjord nearby with no fish farms. The objective of this study was to clarify whether the physiology or taste of saithe near fish farms differs from saithe in areas with no or limited influence from fish farms. The fish collected near farms were larger than those from control sites of the same age and had been eating pellets. Analysis of fatty acid composition of the fillet indicated that fish collected near the farms had fatty acid profiles that resembled that of pellets more than fish from reference sites. The results suggest that there were individual differences in the time spent near cages for saithe collected near the farm, and that saithe collected at the reference area in the same fjord had also been eating pellets, i.e. had visited at least one of the fish farms in the fjord. In a sensory test, the test panel found that saithe from the fjord without fish farms tasted better than saithe collected near the cages. The test panel found no clear differences in taste between saithe collected near the fish farm and saithe from the same fjord, but 6 km from the nearest fish farm.