In vitro plantlet formation from embryonic explants of eastern white cedar (Thuja occidentalis L.)

A protocol is described for the in vitro production of plantlets from embryonic explants of eastern white cedar (Thuja occidentalis L.). Bud induction was optimal when embryonic explants were cultured for 20-25 days on half-strength Quoirin and Le Poivre mineral salts containing equimolar concentrations (10(-6) M) of N(6)-benzyladenine and 2-isopentyl adenine. Bud development was achieved in phytohormone-free medium in the presence of activated charcoal. Maximal shoot elongation occurred on half-strength Quoirin and Le Poivre salts, whereas shoot multiplication was optimal on half-strength Bornman's MCM salts in the presence of cytokinin. Hardened shoots, dipped in commercial rooting powder containing indole-3-butyric acid, rooted optimally in mist under non-sterile greenhouse conditions. Both rooting and subsequent plantlet growth was best when Redi-Earth((R)) was used as a substrate. Over 250 plantlets per embryo can be produced annually by this technique.     

Ecological interactions between fungi, other biota and forest litter composition in a unique Scottish woodland

The composition of forest litter and understorey layer, andfungal biomass (in terms of ergosterol) were measured in eightsubplots over a winter–spring period (January to April).The sampling site was positioned in a range of woodland habitats(variously dominated by beech, Fagus sylvatica; birch, Betulapendula x pubescens, and oak Quercus petraea) and a clear areacovered with grass (dominated by Holcus lanatus). The resultswere analysed together with data on bacteria and microinvertebratesavailable from parallel research. Levels of ergosterol in individualsubplots ranged between 50 and 160 µg g^–1 DW. Fungalbiomass decreased in March, and then increased significantlyin April. Stepwise regression models for ergosterol indicatedpositive relationships with moisture content (February), bacteria(all but February and March), flagellates (February) and plant-feedingnematodes and flies (January, overall). The relationships withroots, seeds, the collective variable ‘other microinvertebrates’(all March), amoebae (February) and fragments (March, overall)were negative, while the relationship between fungi- and microbial-feedingnematodes changed sign between February (–) and March(+). Results of analysis of covariance for fungal ergosterolwere significant only for January and the combined dataset.In January, fungi were shown to be significantly related toamoebae, bacteria and a collembolan Folsomia candida, whilethe only significant predictor returned by the overall modelwas bacteria. Correlation analysis confirmed some effects alreadynoted, and revealed a number of further interactions. The resultshighlighted the complexity of factors influencing temporal dynamicsand spatial variability of fungal biomass in forest litter.Most of the registered interactions appeared to be transient,and this should be taken into account while interpreting environmentalobservations. Interpretation of the specific relationships isgiven and implications for further research and overall ecosystemfunctioning are discussed.     

A study of the bonding forces between the epithelial cells surrounding the resin canals of slash pine (Pinus elliottii Engelm.) holocellulose

Summary Although the bonds between the cellular components of pine epithelia (isolated from holocellulose) can be strengthened by the addition of multivalent ions such as those of calcium, iron, uranium, etc., it was observed that these bonds are only weakened and not destroyed by the removal of the ions from the tissues. Reagents, such as KOH and K₂SO₄, which do not yield soluble calcium salts, do not cause a discernible weakening of the bonds between the cellulose components of epithelia pretreated with Ca ion. Washing the weakened epithelia with water causes them to swell. The separation of epithelial cells occurs only after the application of mechanical forces to the tissues. Electron microscopic examination of the swollen and unswollen tissues shows that fibrils and lamellae extend between the individual cells which must be destroyed by mechanical action before cell separation can occur. The swelling of the tissues by washing after removal of multivalent ions was attributed to the expansion of electrical double layers and to the increasing osmotic pressure from the increasing Donnan potential, while the difference in swelling as a result of HCl treatment vs. KCl and K₄Fe(CN)₆ was attributed to differences in charge development and to hydrogen bonding between acidic components. The restrengthening of the tissue as a result of the readdition of multivalent ion was attributed to the collapse of the electrical double layers and the reduction of the Donnan potential, with the re-forming of bonds between the components of the epithelia. Evidence suggesting that multivalent ions actually participate in bonding was obtained and although the nature of the bonding cannot be determined, both salt formation and complex formation between the ions and the components of the tissues is suggested.     

Assessment of the combination of spinosad and milbemycin oxime in preventing the development of canine Angiostrongylus vasorum infections

Angiostrongylus vasorum is an increasingly reported parasite in Europe that develops in dogs after ingestion of infective third stage larvae (L3) that reside in gastropod molluscs which are needed to complete the parasite's life-cycle. Infection can produce a diversity of clinical signs, determined by involvement of the respiratory, neurological, and/or coagulation system, with a likely fatal outcome in the absence of treatment. Few drugs have been shown to reliably prevent infection, and data on treatment of infections is limited. A controlled, randomized, partially blinded laboratory study was therefore executed to evaluate the efficacy and safety of a combination tablet of spinosad/milbemycin oxime in dogs inoculated with approximately 250 A. vasorum L3. Sixteen healthy nematode free adult dogs were randomly allocated to two study groups of 8 dogs each. Thirty days post inoculation (dpi) all dogs in the fed state were treated: dogs in group B were treated with spinosad and milbemycin oxime at the dose rates of 45–60 mg/kg and 0.75–1.0 mg/kg bodyweight, respectively, approximately the lower half portion of the expected full unit dose range; dogs in group A were treated with placebo tablets. All dogs were euthanized and necropsied 56–58 dpi. The heart and lungs were examined to determine the presence of A. vasorum. All placebo group dogs were infected at necropsy with counts ranging from 22 to 98 adult worms and a geometric mean worm count of 55.2. In contrast, the geometric mean worm count in the spinosad/milbemycin oxime group was 0.7 with worm numbers ranging from 0 to 8. The results of this study demonstrate that a single treatment with the tablet combination of spinosad and milbemycin oxime administered 30 dpi provided 98.8% preventive efficacy against development of adult A. vasorum infections. Monthly treatments with spinosad and milbemycin oxime have the potential to prevent the establishment of infections with A. vasorum in dogs.     

The Post‐Cervical Insemination does not Impair the Reproductive Performance of Primiparous Sows

The study evaluated the reproductive performance of primiparous sows submitted to post‐cervical insemination (PCAI) compared with cervical artificial insemination (CAI). Difficulty with catheter introduction, the occurrence of bleeding or semen backflow during insemination, and volume and sperm cell backflow up to 60 min after insemination were also evaluated. Sows were homogenously distributed, according to body weight loss in lactation, lactation length, weaned piglets, weaning‐to‐oestrus interval and total born in previous farrowing, in two treatments: PCAI (n = 165) with 1.5 × 10⁹ sperm cells in 45 ml (2.4 ± 0.04 doses per sow) and CAI (n = 165) with 3 × 10⁹ sperm cells in 90 ml (2.5 ± 0.04 doses per sow). During PCAI, sows were inseminated in the absence of boars. Transabdominal real‐time ultrasonography was performed at oestrus onset, immediately before the first insemination and at 24 h after last insemination. There was no difference (P > 0.05) between treatments in farrowing rate (91.5% × 89.1%) and litter size (12.5 × 11.9 piglets born, respectively for PCAI and CAI sows). Successful passage of the intrauterine catheter in all the inseminations was possible in 86.8% (165/190) of sows initially allocated to PCAI treatment. Difficulty of introducing the catheter in at least one insemination did not affect the reproductive performance of PCAI sows (P > 0.05). Bleeding during insemination did not affect (P > 0.05) the farrowing rate in both treatments, but litter size was reduced in CAI and PCAI sows (P ≤ 0.06). Percentage of spermatozoa present in backflow within 1 h after insemination was greater in CAI than PCAI sows (P < 0.01). More than 85% of primiparous sows can be successfully post‐cervical inseminated with doses containing 1.5 × 10⁹ sperm cells in the absence of the boar during insemination without impairing the reproductive performance.     

Effect of Breeding Activity on the Microflora of the External Genitalia and in the Semen of Stallions,and the Relationship Between Micro‐organisms on the Skin and on the External Genitalia

A possible role of breeding activities in the composition of the microbial population in stallions' external genitalia (EG) and the relationship between micro‐organisms colonizing the skin of the abdomen and the ones colonizing the EG have not been studied. In experiment 1, EG microbiological samples were collected from 41 stallions used for both natural cover and semen collection (BST) and from 18 non‐breeding stallions (NBST). A higher (p < 0.05) frequency of isolation of potentially pathogenic species was found for BST. Age did not influence number of micro‐organism species isolated both in BST and NBST. In experiment 2, the microbial content of the EG and semen was compared in 23 BST. Most micro‐organisms isolated from the EG were present in semen, albeit with a numerically lower prevalence. In 7 stallions, six microbial species isolated from semen were absent from the EG cultures, suggesting contamination by the operator. In experiment 3, a numerically higher number of micro‐organism species was isolated from the EG of 31 stallions, than from their skin of the ventral abdomen in contact with the penis or from the skin of the thorax. With the sole exception of Escherichia coli, potentially pathogenic bacteria were only isolated from the EG but not from the skin. Results suggest that breeding activity increased the number of species colonizing the EG; most species isolated from the EG were also found in semen even if with a lower frequency, and additional semen contamination seemed to occur during its manipulation. Many micro‐organism species of the skin were also isolated from the penis, but independently of being or not in contact with the penis, skin did not seem to provide an adequate environment for the growth of potentially pathogenic bacteria that were isolated from EG, with the sole exception for E. coli.     

The effect of dietary protein on the growth and survival of the shrimp, Penaeus monodon in outdoor tanks

The comparative effect of reducing the protein content of formulated feed on the growth and survival of black tiger shrimp, Penaeus monodon, and on water quality was tested in outdoor tanks. Three diets, 300, 350 and 400 g kg^?1 crude protein (CP), were fed to P. monodon (3.1 g animals, 25 animals per m²) in each of eight replicated outdoor 2500 L tanks in an 8‐week trial. There was no statistical difference (P > 0.05) in shrimp growth rate (1.34–1.50 g week^?1), survival, or final biomass between the treatments. However, when tanks with lower survival were removed from the analysis (<60 and <80% were tested), shrimp growth rate was statistically higher (P < 0.05) in the 350 and 400 g kg^?1 CP diets than in the 300 g kg^?1 CP diet treatment. There were no differences in the nutritional condition of shrimp between treatments, as determined by moisture and protein content, and lipid content of the digestive gland. Using ¹⁵N‐nitrogen isotope tracers, it was determined that shrimp were consuming natural biota, although these were unlikely to have contributed substantially to their nutrition. Total nitrogen (TN) concentrations in the water column increased over the eight week experiment and were statistically different (P < 0.001) between treatments (3.60, 5.17 and 6.45 mg L^?1 in the 300, 350 and 400 g kg^?1 CP treatments, respectively). Concentrations of dissolved organic nitrogen (DON) were also statistically different between treatments and made up 35–40% of the TN in the water column. Concentrations of total ammoniacal nitrogen (TAN) and oxides of nitrogen, and fluorescence were not statistically different between treatments but there was a trend of higher concentrations in treatments with higher protein levels. There was no difference in sediment nutrients between treatments. This study has shown that there is scope to reduce the protein content of P. monodon diets but only by 5–10%. However, further validation of these results in commercial ponds is needed. Reducing the feed protein content may result in cost savings and also has the advantage of improving water quality and reducing nitrogen discharge.     

Identification of Mycobacterium spp. isolated from snakehead, Channa striata (Fowler), and Siamese fighting fish, Betta splendens (Regan), using polymerase chain reaction–reverse cross blot hybridization (PCR–RCBH)

A variety of methods have been used to identify Mycobacterium spp. isolated from snakehead and Siamese fighting fish, including biochemistry, mycolic acid profiles and antibody-based methods. However, these methods are unable to differentiate between different species of Mycobacterium . Polymerase chain reaction (PCR) followed by reverse cross blot hybridization (RCBH) was adapted in this study to speciate aquatic mycobacteria. The method was highly specific for Mycobacterium spp. and identified the bacteria to species level with a detection limit of 100 fg DNA, equivalent to 20 mycobacteria. Twenty-nine isolates previously collected and cultured from Siamese fighting fish (10 isolates) and snakehead (19 isolates) during outbreaks of mycobacteriosis were analysed using PCR–RCBH. Six of the Siamese fighting fish isolates and nine of the snakehead isolates were identified as Mycobacterium fortuitum , while the remainder were classified as M. marinum . Notably, two isolates recovered from snakehead and Siamese fighting fish, previously identified as M. poriferae and M. piscicida , respectively, were confirmed to be M. fortuitum .