PI4P and PI(4,5)P2 are essential but independent lipid determinants of membrane identity

The quantitatively minor phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P(2)] fulfills many cellular functions in the plasma membrane (PM), whereas its synthetic precursor, phosphatidylinositol 4-phosphate (PI4P), has no assigned PM roles apart from PI(4,5)P(2) synthesis. We used a combination of pharmacological and chemical genetic approaches to probe the function of PM PI4P, most of which was not required for the synthesis or functions of PI(4,5)P(2). However, depletion of both lipids was required to prevent PM targeting of proteins that interact with acidic lipids or activation of the transient receptor potential vanilloid 1 cation channel. Therefore, PI4P contributes to the pool of polyanionic lipids that define plasma membrane identity and to some functions previously attributed specifically to PI(4,5)P(2), which may be fulfilled by a more general polyanionic lipid requirement.     

K‐Fertilization under different Agroecological conditions studied with maize

In Hungary the AL‐method using ammonium lactate acetic acid as extradants has been widely used for serial analysis of soil potassium and phosphorous available to plants in the last 30 y. Because of the considerable increase of the price of fertilizers and decline of the economic situation, there is a pressing need for cost‐effective fertilizer use. Recently this highlighted the importance of nutrient recommendations based on soil analyses. Small plot long‐term field fertilization experiments are the best means to quantify the efFect or the efficiency of K‐fertilization. Long‐term data sets can reveal on which soil type which crop responds most favourably to the different fertilizer doses and nutrient ratios under different, long‐term balanced soil nutrient levels. Since the experimental data of the National Long‐Term Field Trials reflect the agro‐ecological conditions of nine experimental sites representing different soil types of Hungary, these experiments provide excellent opportunity to study this topic. The trials were set up in 1967 with 20 fertilization treatments including three increasing N‐and P‐levels each, two K‐levels and their different combinations. From the experimental data of great number only those of four sites will be shown. Winter wheat, maize, and pea have been grown as test plants. In the experiment selected for presentation, maize had statistically proved responses to K‐fertilization. The efficiency of K‐fertilizer on different soil types shows, however, significant differences, which can not be completely explained with the results of K‐analysis gained with the AL‐method.     

In vitro production of porcine embryos: current status,future perspectives and alternative applications

The pig is considered to be a suitable source of cells and organs for xenotransplants, as well as a transgenic animal to produce specific proteins, given the biological similarities it shares with human beings. However, the in vitro embryo production system in pigs is inefficient compared with those in other mammals, such as cattle or mice. Although numerous modifications have been applied to improve the efficiency of in vitro embryo production systems in pigs, not much progress has been made to overcome the problem of polyspermy, and low developmental ability due to insufficient cytoplasmic abilities of in vitro matured oocytes and improper culture conditions for the in vitro produced embryos. Recent achievements, such as the establishment of chemically defined medium and utilization of ‘zona hardening’ technique, have gained some success. However, further research for the reduction of polyspermy and detrimental effects of the culture systems in pigs is still needed.     

Continuous versus intermittent thermodilution for cardiac output measurement during alveolar recruitment manoeuvres in sheep

ObjectiveTo evaluate interchangeability of a thermodilution based STAT mode continuous cardiac output (CCO) measurement method with bolus thermodilution (BTD).Study designRandomized crossover study.AnimalsTen 9 month old healthy male sheep.MethodsEach sheep was anaesthetized twice for laparoscopy. On one occasion mechanical ventilation was used immediately after anaesthetic induction (IPPV treatment) and on the other occasion the start of IPPV was delayed and two periods of alveolar recruitment manoeuvres were also performed (RM treatment). Cardiac output (CO) was measured simultaneously with both CCO and BTD at 6 time points. Data were analysed using difference versus mean plots. A priori limits of acceptance were set at ±30% of the mean of every paired measurement. If <5% of the data fell outside of these limits (Chi-square test, p < 0.05) the interchangeability of methods was accepted. Proportions of data outside of these limits were also compared between treatments (Fisher's test, p < 0.05). Cardiac output data from each treatment and measurement method were also analyzed separately with one-factorial anova and Bonferroni test (p < 0.05).ResultsA total of 119 measurements were obtained. Cardiac output ranged from 1.9 to 10.4 L minute^?1 (CCO) and from 1.1 to 9.8 L minute^?1 (BTD). The bias and limits of agreement were 0.5 ± 1.9 L minute^?1. More than 5% of all data fell outside of the limits of acceptance (24/119), and a larger proportion fell outside of these limits in the RM (20/59) compared to the IPPV treatment (4/60). The Bonferroni test detected significant decreases of CO over time in both treatments when measured with BTD but not with CCO.Conclusions and clinical relevanceThe STAT mode CCO method is not interchangeable with BTD during acute haemodynamic changes caused by recruitment manoeuvres, thus the results of STAT mode CCO should be interpreted with caution because decreases in CO may not be detected.     

Cytoskeletal abnormalities in relation with meiotic competence and ageing in porcine and bovine oocytes during in vitro maturation

We investigated the frequencies of cytoskeletal anomalies in metaphase-II (M-II) and incompetent [arrested at an immature metaphase (IM) stage] porcine and bovine oocytes during in vitro maturation (IVM) in relation with ageing by immunostaining and confocal microscopy. In porcine oocytes, meiotic arrest at the IM stage was associated with abnormalities of cortical actin but not with abnormal spindles. Prolongation of IVM culture to 52 h did not affect microfilament and spindle abnormalities, but reduced the microfilament-rich area overlaying the spindle. Meiotic arrest of bovine oocytes at the IM stage was associated with degenerations of microfilaments, and the frequencies of abnormal spindles were also higher than those of M-II oocytes. Ageing of bovine oocytes (IVM for 30 h) did not affect cortical microfilaments but increased the frequency of spindle alterations in both M-II and IM bovine oocytes. These results suggest that, in both species, altered ability of oocytes to polymerize F-actin might be a possible reason for the failure of polar body extrusion during IVM. Also, there seem to be differences between the two species in the sensitivity of oocytes to suffer ageing-related spindle damages.     

In-straw cryoprotectant dilution for bovine embryos vitrified using Cryotop

The aim of this study was to develop an in-straw dilution method suitable for 1-step bovine embryo transfer of vitrified embryos using the Cryotop vitrification-straw dilution (CVSD) method. The development of embryos vitrified using the CVSD method was compared with those of embryos cryopreserved using in-straw vitrification-dilution (ISVD) and conventional slow freezing, outside dilution of straw (SFODS) methods. In Experiment 1, in vitro-produced (IVP) embryos cryopreserved using the CVSD method were diluted, warmed and exposed to the dilution solution at various times. When vitrified IVP embryos were exposed to the dilution solution for 30 min after warming, the rates of embryos developing to the hatched blastocyst stage after 72 h of culture (62.0-72.5%) were significantly lower (P<0.05) than those of embryos exposed to the solution for 5 and 10 min (82.4-94.3%), irrespective of supplementation with 0.3 M sucrose in the dilution solution. In Experiment 2, the rate of embryos developing to the hatching blastocyst stage after 48 h of culture in IVP embryos cryopreserved using the SFODS method (75.0%) was significantly (P<0.05) lower than those of embryos cryopreserved using the CVSD and ISVD methods (93.2 and 97.3%, respectively). In Experiment 3, when in vivo-produced embryos that had been cryopreserved using the CVSD, ISVD and SFODS methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception and delivery rates among groups. In Experiment 4, when IVP embryos derived from oocytes collected by ovum pick-up that had been cryopreserved using the CVSD and ISVD methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception rates among groups. Our results indicate that this simplified regimen of warming and diluting Cryotop-vitrified embryos may enable 1-step bovine embryo transfer without the requirement of a microscope or other laboratory equipment.     

The effect of resveratrol on the developmental competence of porcine oocytes vitrified at germinal vesicle stage

We tested the effects of resveratrol both as a pre‐treatment and as a recovery treatment after warming during in vitro maturation (IVM) on the viability and developmental competence of porcine oocytes vitrified at the germinal vesicle stage. Pre‐treatment before vitrification of oocytes for 3 hr with 2 μM resveratrol did not affect survival, oocyte maturation and embryo developmental competence to the blastocyst stage after parthenogenetic activation. However, supplementation of the medium with resveratrol during subsequent IVM after vitrification and warming significantly improved the ability of surviving oocytes to develop to the blastocyst stage, and this effect was observed only on vitrified, but not on non‐vitrified oocytes. The intracellular levels of glutathione and hydrogen peroxide in oocytes were not affected by vitrification and resveratrol treatment. Also, there was no significant difference in the occurrence of apoptosis measured by annexin V binding between vitrified and non‐vitrified oocytes, regardless of the resveratrol treatment. In conclusion, resveratrol did not prevent the cellular damages in immature porcine oocytes during vitrification; however, when added to the IVM medium, it specifically improved the developmental competence of vitrified oocytes. Further research will be necessary to clarify the mechanisms of action of resveratrol on the recovery of vitrified oocytes from vitrification‐related damages.     

Optimization of the in vitro fertilization protocol for frozen epididymal sperm with low fertilization ability in Ban—A native Vietnamese pigs

The aim of the present study was to improve the penetration during in vitro fertilization (IVF) of a frozen lot of epididymal sperm with a notoriously low fertilization ability of a Ban boar which is a native Vietnamese breed by optimizing different parameters of the IVF system. In Experiment 1, we determined that Pig‐fertilization medium was superior medium to Tyrode's albumin lactate pyruvate‐polyvinyl alcohol medium for IVF and defined the optimum the sperm concentration (1 × 10⁶ sperm/ml). In Experiment 2, we clarified that partial removal of cumulus cells from cumulus‐oocyte complexes by hyaluronidase treatment before IVF enhances sperm penetration, whereas complete cumulus removal reduces penetration. Finally, in Experiment 3 the elevation of concentration of caffeine in Pig‐fertilization medium from 2 to 5 mmol/L and the prolongation of the co‐culture of gametes from 3 to 5 hr significantly increased the total penetration rate from 15.2% to over 50%. In conclusion, the combination of partial oocyte denudation, an elevated caffeine concentration in Pig‐fertilization medium and an extended interval of IVF with using an optimized sperm concentration was a potent way to improve the fertilization results for a frozen epididymal Ban sperm lot with low fertility.     

Cryopreservation of immature oocytes of the indigeneous Vietnamese Ban Pig

We report the cryopreservation of oocytes from Ban miniature pigs which are endemic in Vietnam. Immature cumulus‐oocyte complexes were collected from antral follicles of 7–8 mo old female cyclic Ban pigs and vitrified in micro‐drops. Oocyte morphology, lipid content, post‐warming survival, nuclear maturation, and embryo development were compared to those of oocytes from commercially slaughtered Landrace × Large white hybrid pigs. The size of oocytes in the two breeds was similar. However, significantly lower amounts of intracellular lipid were detected in Ban oocytes. There was no difference (p > 0.05) between Ban and Landrace × Large white oocytes in percentages of post‐warming survival (93.1 ± 3.4% vs. 70.7 ± 16.7%, respectively) and nuclear maturation after in vitro maturation (80.4 ± 5.1% vs. 90.0 ± 1.3% respectively). Similarly, cleavage (30.8 ± 7.8% vs. 10.3 ± 6.1%, respectively) and blastocyst development rates (9.4 ± 5.0% vs. 0.79 ± 0.79, respectively) were not different (p > 0.05) between vitrified Ban and Landrace × Large white oocytes after in vitro fertilization and embryo culture. In conclusion, high survival and maturation rates were achieved after vitrification of immature Ban oocytes and their cryo‐tolerance was similar to that of Landrace × Large white oocytes, despite the difference in lipid content. We succeeded to generate reasonable rates of blastocysts from vitrified Ban oocytes by in vitro fertilization.     

Soil fertility in a long‐term fertilizer trial with different tillage systems

The presented results originate from a field experiment established in 1972 on an Eutric cambisol with two main factors: soil tillage (conventional‐, reduced‐, and no‐tillage) and NPK fertilization. The test plants were maize and winter wheat in two years rotation.

The long‐term soil fertility without and with optimum fertilization, the influence of fertilization, tillage and crop sequence on grain yields, the organic carbon content (C_org) and the nitrate infiltration are discussed.

In the course of years without any NPK fertilization grain yields of maize and winter wheat decreased significantly and reached a minimum level which was modified however by the actual climatic conditions. The analogous yield level of optimum NPK fertilization at maize showed a growing tendence while at wheat it remained mostly constant.

The method of soil tillage influenced grain yield of winter wheat to a lesser extent than the yield of maize. Grain yields of maize and winter wheat were consistently lower with no‐till as compared to reduced or conventional tillage, however the differences with w. wheat were much smaller. The effect of tillage was especially high at N₀ P₀ K₀. Crop rotation had a positive effect on the yields of maize. For winter wheat at N₀ P₀ K₀ oneself was the better forecrop, while at optimum N and PK maize performed a little better. C_orgcontent of soil slightly increased in the course of 25 years not only on the fertilized plots but on the nil plots too. Increasing N‐doses showed only a little effect on the C_org. There was a little positive effect of no‐till on C_org content of soil as well as compared fall ploughing. Soil tillage did not much influence the total amount of nitrate in the soil profile. The distribution of nitrate‐N in the soil profile was more affected by the actual climatical circumstances than by the system of tillage. However big nitrate accumulations were found in the subsoil according to different soil tillage systems at some other times, as well as lack of it, which suppose the possibility of a relative quick nitrate infiltration.