Spermatophore cryopreservation and artificial insemination of black tiger shrimp, Penaeus monodon (Fabricius)

To develop an appropriate cryopreservation protocol for spermatophores of black tiger shrimp, Penaeus monodon, three cryoprotectants (dimethyl sulphoxide (DMSO), methanol (MeOH) and ethylene glycol (EG)) at two concentrations (5% and 10%) were examined. Artificial implantation of spermatophores was also carried out to assess the fertilizing ability of fresh and post‐thaw spermatophores. Spermatophores were collected during consecutive regenerations (15‐day intervals) and assessed for qualitative and quantitative changes and also for fertilizing ability by implantation. The mean fertilization rate for artificial insemination using post‐thaw spermatophore was 79.9±3.7%, lower than the fertilization rates observed for artificial implantation using fresh spermatophore and natural mating. Mean hatch rates for fresh spermatophore, frozen‐thawed spermatophore and natural mating were 88.8±0.6%, 87.8±0.4% and 88.3±0.5%, respectively; and there was no difference among the three groups. The mean fertilization rate of spermatophores collected during the first stripping was higher (90.6±0.6) than during the second stripping (85.7±2.6), but the mean hatch rate was not different between the two strippings. The highest mean sperm viability (79.7±0.4%) was obtained from DMSO (5%), with no survival observed in the 10% MeOH treatment. Spermatophore weight, total sperm count and percentage of abnormal sperm were not different between spermatophores collected at the first and second stripping. This is the first study to report high fertilization and hatch rates from cryopreserved spermatophore using artificial implantation of spermatophore before spawning.     

Changes of Ca2＋ activated potassium channels and cellular-proliferation in autogenous vein grafts

AIM: To investigate changes of Ca^2＋ activated potassium channels (K_Ca) in autogenous vein grafts. METHODS: The contraction of venous ring was measured by means of perfusion in vitro. The intimal proliferation and proliferation of cultured smooth muscle cells (vascular smooth muscle cells, VSMCs) were observed by the means of computerised image analysis and MTT method, respectively. Furthermore, whole cell mode of patch clamp was used to record K_Ca of VSMCs isolated from autogenous vein grafts. RESULTS: 1 week after transplantation there were no significant differences of contraction and intimal relative thickness between autogenous vein grafts and control. Contraction and intimal relative thickness of autogenous vein graft were significantly increased 2 weeks after transplantation (P＜0.05, n=8 vs control), and they were more enhanced 4 weeks after vein transplantation (P＜0.01, n=8 vs control). TEA (blocker of Ca^2＋ activated potassium channels) increased MTT A₄₉₀ value of VSMCs from femoral vein in a dose-dependent manner (P＜0.05, n=8). K_Ca current density was significantly attenuated in VSMCs from autogenous vein grafts 1-4 weeks after transplantation (P＜0.05, n=5). CONCLUSION: K_Ca was inhibited in autogenous vein graft, which accounted for vasospasm and intimal proliferation.     

Bovine blood neutrophil acyloxyacyl hydrolase (AOAH) activity during endotoxin and coliform mastitis

The dynamics of blood neutrophil acyloxyacyl hydrolase (AOAH) activity, the appearance of endotoxin (lipopolysaccharide, LPS) in blood and the role of blood neutrophil AOAH in the severity of Escherichia coli and endotoxin mastitis were investigated in early postpartum dairy cows experimentally challenged with either endotoxin (n = 6) or E. coli (n = 6). The AOAH activity of blood neutrophils started to decrease significantly at post challenge hours (PCH) 6-24 and 12-24 in the endotoxin and E. coli-challenged groups, respectively; it returned to pre-challenged values at PCH 48 in both endotoxin- and E. coli-challenged groups. The cows were classified as moderate and severe responders according to milk production loss in the non-challenged quarters at PCH 48. There were no severe responders in the endotoxin-challenged group. In the E. coli-challenged group, only 1 severe responder was identified. The pre-challenge neutrophil AOAH activity of the severe responder was approximately 30% lower than that of moderate responders. No LPS was detected in the plasma of endotoxin-challenged cows; neither was it found in the plasma of moderate responders in the E. coli-challenged group at any PCH. However, at PCH 6, a remarkable amount of LPS was detected in the plasma of the severe responder from the E. coli-challenged group. Furthermore, neutrophil AOAH activity was increased by approximately 70% in the severe responder at PCH 6, but it increased by only approximately 15% in moderate responders. This was followed by a decreased neutrophil AOAH activity at PCH 12-24 and 24-72 in moderate and severe responders, respectively; the decreased AOAH activity at those PCH was more pronounced in the severe responder. The pronounced decreased neutrophil AOAH activity during mastitis often coincided with extreme leukopenia, neutropenia and a maximal number of immature neutrophils in the blood. Our results demonstrate that a decrease in neutrophil AOAH activity results in the appearance of LPS in the blood, and low blood neutrophil deacylation activity could be considered as a risk factor for severe clinical coliform mastitis.     

Electrotrawling: a promising alternative fishing technique warranting further exploration

In trawl fisheries, beam trawls with tickler chains, chain mats or bobbin ropes are used to target flatfish or shrimp. High fuel consumption, seabed disturbance and high discard rates are well‐known disadvantages of this fishing technique. These shortcomings are increasingly gaining international public and political attention, especially with the upcoming discard ban in Europe. The most promising alternative fishing technique meeting both the fisherman's aspirations, and the need for ecological progress is pulse fishing with electrotrawls. Here, the mechanical stimulation by tickler chains or bobbins is replaced by electrical stimulation resulting in reduced bottom contact, fuel costs and discards. Although a significant amount of research has been done on electrotrawls and their impact on marine organisms, most data were published in very diverse sources ranging from local non‐peer‐reviewed reports with a limited distribution to highly consulted international peer‐reviewed journals. Therefore, there is a clear need for a comprehensive yet concise and critical overview, covering and summarizing all these data and making these available for the scientific community. This article aims to meet the above goals by discussing the working principle of electric fields, the history of electrotrawls and their current application in the North Sea and impact on marine organisms. It is concluded by elaborating on the opportunities and challenges for the further implementation of this alternative fishing technique.     

Quantitative PCR monitoring of the effect of azoxystrobin treatments on Mycosphaerella graminicola epidemics in the field

Quantitative PCR and visual monitoring of Mycosphaerella graminicola epidemics were performed to investigate the effect of curative and preventative applications of azoxystrobin in wheat field crops. A non-systemic protectant and a systemic curative fungicide, chlorothalonil and epoxiconazole, respectively, were used as references. PCR diagnosis detected leaf infection by M graminicola 3 weeks before symptom appearance, thereby allowing a clear distinction between curative and preventative treatments. When applied 1 week after the beginning of infection, azoxystrobin curative activity was intermediate between chlorothalonil (low effect) and epoxiconazole. When applied preventatively, none of the fungicides completely prevented leaf infection. There was some indication that azoxystrobin preventative treatments may delay fungal DNA increase more than epoxiconazole at the beginning of leaf infection. Both curative and preventative treatments increased the time lapse between the earliest PCR detection and the measurement of a 10% necrotic leaf area. Azoxystrobin only slightly decreased the speed of necrotic area increase compared with epoxiconazole. Hence, azoxystrobin activity toward M graminicola mainly resides in lengthening the time lapse between the earliest PCR detection and the measurement of a 10% necrotic leaf area. Information generated in this way is useful for optimal positioning of azoxystrobin treatments on M graminicola.     

Seasonal variation of inorganic nitrogen and net mineralization in a salt marsh ecosystem

Inorganic nitrogen pools and net mineralization were estimated in three sites of a Tagus estuary salt marsh in Portugal throughout 1 year. Ammonium (NH₄ ⁺) was the major form of inorganic nitrogen found in the salt marsh soil. Extractable NH₄ ⁺ concentrations showed a marked seasonal pattern with a concentration peak during the hotter months of July/August. The great majority (>99%) of the total nitrogen in the soil was found in sedimented organic matter, not readily available for plant uptake. Net nitrogen mineralization, determined using a field incubation method, showed a peak during the months of June/July which resulted in an increase on nitrogen availability. With the exception of the lower salt marsh, estimated rates of in situ net nitrogen mineralization in the soil during summer were well related to the increase in plant aboveground biomass and plant nitrogen pools, indicating that the process is an important source of available nitrogen for plant uptake and growth. Annual net nitrogen mineralization ranged between 2.4 and 4.5gNm^–2yr^{– 1} being significantly higher for the lower salt marsh site. Rates of net nitrogen mineralization were relatively low during most of the year with a particularly active period from June to August, possibly due to an effect of temperature on soil microbial activity.     

Spatial pattern of the density of Armillaria epiphytic rhizomorphs on tree collar in an oak stand

The density of Armillaria gallica epiphytic rhizomorphs on tree collar was mapped in a young stand of pedunculate oaks (Quercus robur). A quick method to estimate the rhizomorph density was developed, investigating 19 trees, and was used in the subsequent study. The method consists of measuring the frequency of rhizomorphs present on a grid applied to an exposed small area of the tree collar. The pattern in tree collar rhizomorph density was closely related to the pattern of stump colonization by Armillaria and stumps were therefore important as inoculum sources. Rhizomorph density on tree collar showed a strong aggregated spatial pattern which could not be explained by variability in the soil characteristics, in particular waterlogging, or by a spatial pattern in tree vigour. Moreover, rhizomorph density on tree collar did not depend on their dominance status.     

Co‐expression of PACAP with VIP,SP and CGRP in the Porcine Nodose Ganglion Sensory Neurons

Our previous study revealed the expression of substance P (SP) and calcitonin gene‐related peptide (CGRP) in sensory distal ganglion of the vagus (nodose ganglion) neurons in the pig. As these neuropeptides may be involved in nociception, the goal of these investigations was to determine possible expression of vasoactive intestinal polypeptide (VIP), SP and CGRP in the pituitary adenylate cyclase‐activating polypeptide‐immunoreactive (PACAP‐IR) porcine nodose perikarya. Co‐expression of these substances was examined using a double‐labelling immunofluorescence technique. To reveal the ganglionic cell bodies, the pan‐neuronal marker protein gene product 9.5 (PGP 9.5) was used. Quantitative analysis of the neurons revealed that 67.25% of the PGP 9.5+ somata in the right‐side ganglion and 66.5% in the left side, respectively, co‐expressed PACAP‐IR. Moreover, 60.6% of the PACAP‐IR cells in the right‐side ganglion and 62.1% in the left, respectively, co‐expressed VIP. SP‐IR was observed in 52.2 and 39.9% of the right and left ganglia, respectively. CGRP was found in 27.7 and 34.1% of the right and left distal ganglion of the vagus, respectively. High level of co‐expression of PACAP with VIP, SP and CGRP in the distal ganglia of the vagus sensory perikarya directly implicates studied peptides in their functional interaction during nociceptive vagal transduction.