Evaluation of embryonic age and the effects of different proteases on the isolation and primary culture of chicken intestinal epithelial cells in vitro

The present study evaluates the effects of embryonic age and proteolytic enzymes on the isolation and primary culture of chicken enterocyte and to establish an effective technique for chicken intestinal epithelial cell (IEC) cultivation. Fourteen‐day‐old, 16‐day‐old and 18‐day‐old embryos (average weight: 52.23 ± 0.76 g, 50.86 ± 0.99 g, 48.98 ± 1.03 g) were the source for preparation of enterocyte culture, and trypsin‐ethylene diamine tetraacetic acid, collagenase, thermolysin and combination of collagenase and thermolysin were used for digestion medium. Optimal culture protocols were determined by qualitative assays of proliferation. Cells isolated by using 14‐day‐old embryo and collagenase obtain the best attachment and growth in culture, and the production of continuously growing IEC cultures. Thus, we conclude that the use of collagenase as a dissociating enzyme and 14‐day‐old embryo as a source can be advantageously applied to the isolation of chicken IEC and this method may be useful for various applications and basic studies of the intestinal tract concerning such objects as physiology, immunology and toxicology.     

Evaluating the probiotic potential and adhesion characteristics of Bacillus spp. isolated from the intestine of Rhynchocypris lagowskii Dybowski

Probiotics have been widely used in aquaculture worldwide especially in China as antibiotics have been banned in aquaculture. In the present study, 27 potential strains were isolated from Rhynchocypris lagowskii Dybowski intestinal tract towards the assessment of their probiotic potential. Two potential probiotics were finally screened from the 27 candidate strains according to the results of enzyme-producing ability, in vitro against pathogens ability and antibiotic sensitivity test. The 2 potential probiotics were identified and confirmed on the basis of their colony morphology, biochemical characteristics, and 16S rRNA sequencing. The probiotic strains, LSG2-3–2 and LSG3-6, were identified to be Bacillus methylotrophicus and B. tequilensis, respectively. Further studies showed that LSG2-3–2 and LSG3-6 had excellent tolerance to high temperature (80℃), low pH (3–5), bile salts (0.3%), intestinal juice (10%), and gastric juice (0.5%). The adhesion rates of LSG2-3–2 and LSG3-6 in the in vitro intestinal mucosal adhesion model were 17.74% and 24.04%, respectively. Analysis of their bacteria surface adhesive proteins revealed that the lectins on the bacterial surface of LSG2-3–2 and LSG3-6 were mainly protein and glycoprotein properties, respectively. The adhesion receptor components in the mucus proteins of the two strains were all protein properties. The results of the inhibitory adhesion test indicated that LSG3-6 had a higher inhibitory effect on Aeromonas hydrophila and LSG2-3–2 had a better inhibitory effect on A. veronii. The biosafety assay confirmed that the isolates were not pathogenic to the host fish. Based on the presently observed characteristic features, it can be concluded that LSG2-3–2 (B. methylotrophicus) and LSG3-6 (B. tequilensis) retrieved from the digestive tract of R. lagowskii can be used for the healthy breeding and disease prevention of R. lagowskii.

     

Sesbania aegyptiaca as promising biomass for manufacturing of MDF

Sesbania fiber is a fast-growing wood species that has been investigated for medium-density fiberboard (MDF) production. To assess the possibility of applying the local industrial defibration parameters of sugar-cane bagasse (SCB) on defibration of sesbania, the chemical constituents of unfibrated and defibrated sesbania, as well as their thermal stability and scanning electron micrographs, were estimated. Different preparation variables of MDF, such as density, level of urea-formaldehyde (UF) resin (with 0.19% free-formaldehyde [HCHO]), and pressing time were studied, in comparison with that produced by using SCB fibers. The results showed that most of the tested sesbania-based MDFs have mechanical properties that fulfill the minimum requirements of MDF ANSI standard. Additionally, applying 12% UF and pressing for 240 sec provided sesbania-based MDF with optimum reduction in thickness swelling (reached ～7%). It is important to note that the sesbania-based MDF produced under these conditions is characterized by a lower TS property, than that obtained from SCB, or that reported in standards. The preliminary feasibility study revealed that using sesbania fibers will be an added economical potential for MDF production.     

Karyological analysis of endangered Caspian salmon, Salmo trutta caspius (Kessler, 1877)

The karyotype and chromosomal characteristics of endangered Caspian salmon, Salmo trutta caspius, in the southern part of the Caspian Sea were investigated. From the total of 60 mitotic metaphases achieved from 15 individuals, 15, 9 and 36 metaphases were with the mode <76, 76 and 80 representing 25%, 15% and 60% of metaphases respectively. So, the most common pattern of chromosome number was 80 (36 metaphases, 60%) and the number of diploid chromosomes was thus confirmed as (2n=80) of this subspecies. The karyotype consists of seven metacentric, five submetacentric and 28 telocentric pairs. The karyological parameters of the Caspian salmon, the centromic index, arm ratio, relative length and length variation range of chromosomes, were 0–0.5, 1–∞, 0.011–0.045 and 0.507–2.028 μm respectively. The total length of chromosomes in haploid series was 44.776 μm and fundamental number of chromosome arms was 104. This study may provide the first knowledge on chromosome analysis in Caspian salmon and add basic information useful for its chromosomal manipulations.     

Efficacy of an equal blend of canola oil and poultry fat as an alternate dietary lipid source for Atlantic salmon (Salmo salar L.) in seawater. II: effects on haematology and immunocompetence

This research examined the haematological and immunological responses of quadruplicate groups of juvenile (～400 g initial weight) Atlantic salmon (Salmo salar L.) that had each been fed daily to satiation for 12 weeks one of three high‐energy extruded diets of identical composition except for the supplemental dietary lipid (234.7 g kg^?1) source. The three experimental diets varied in the composition of supplement lipid; diet 1 contained 100% anchovy oil (AO), while diets 2 and 3 replaced 29.8% and 59.7% of the AO (respectively) with a 1:1 blend of canola oil (CO) and poultry fat (PF). Immediately following the feeding trial, a random sample of fish from each diet was sampled for determination of baseline levels of various haematological and immunological parameters. Thereafter, duplicate diet groups were vaccinated (against Listonella anguillarum) and reared on their respective experimental diets for an additional 4 weeks. At that time, the remaining fish were sampled similarly, and the different parameters were measured again. Comparisons between the different diet treatment groups were made before and after vaccination. There were no significant diet treatment effects at either sample time, for haematocrit, differential leucocyte counts, erythrocyte counts, serum hemolytic activity or head kidney leucocyte respiratory burst activity. The fish fed diet 1 however, did show significantly higher post‐vaccination levels of peripheral blood leucocyte respiratory burst activity and higher serum antibody titres against L. anguillarum. The results suggest that the relatively low n‐6/n‐3 fatty acid ratios in the muscle and presumably other tissues of fish fed diet 1, may have resulted in a reduced production of immunocompromising eicosanoids than were produced in fish ingesting the other two diets that were based in part on the different amounts of the CO and PF blend. Long‐term studies are required to confirm this possibility.     

Effect of varying density and water level on the spawning response of African catfish Clarias gariepinus: Implications for seed production

The development of a reliable methodology for spawning of African catfish, Clarias gariepinus without the use of hormone injections would greatly improve the prospects of aquaculture in Africa. Earlier work has shown that it is possible to produce C. gariepinus fingerlings by subjecting the broodfish to a physical stress of reduced water depth and/or increased temperature. The hypothesis that C. gariepinus could be induced to spawn through a combined physical stress of lowered water level and increased stocking density was tested in concrete tanks. Three water levels (25, 50 and 75 cm) and three stocking densities (2, 4 and 6 pairs of broodfish at a 1:1 sex ratio in each hapa) were tested. Water depth in the tanks and brood fish density in the hapas affected spawning success. The percentage of spawning females was significantly higher when broodfish were stocked at 2 and 4 pairs in each hapa at water levels of 25 cm or 50 cm. There was no significant difference in spawning response between the 25 and 50 cm depths while a significant difference was seen between the 75 cm and both 25 and 50 cm depths. The results indicate optimum levels and densities for enhancing spawning success in C. gariepinus.     

Nutritive value of partially dehulled and extruded sunflower meal for post-smolt Atlantic salmon (Salmo salar L.) in sea water

This study determined the digestibility of protein in partially dehulled sunflower meal (SFM) and then, as the main goal, the nutritive value of high‐temperature extruded (≤149°C) partially dehulled SFM (SFM_EX) for post‐smolt Atlantic salmon Salmo salar in sea water. The digestibility study was conducted using the settling column approach (‘Guelph system’) for faeces collection as described by Hajen, Higgs, Beames and Dosanjh. In the nutritive value study, triplicate groups of 50 salmon (mean weight ～116 g) in 4000‐L outdoor fibreglass tanks supplied with 25–40 L min^?1, filtered, oxygenated (dissolved oxygen, 7.0–8.5 mg L^?1), 11–12°C sea water (salinity, 29–31 g L^?1), were fed twice daily to satiation one of five steam‐pelleted dry diets that contained 422 g of digestible protein (DP) kg^?1 and ～16.4 MJ of digestible energy (DE) kg^?1 on a dry weight basis for 84 days. Low‐temperature‐dried anchovy meal (LT‐AM) comprised 68.2% of the basal diet protein whereas in four test diets, SFM_EX progressively replaced up to 33.0% of the DP provided by LT‐AM in the basal diet (SFM_EX≤271 g kg^?1 of dry matter). Sunflower meal had 87.9% DP. Diet treatment did not significantly affect specific growth rate (1.39–1.45% day^?1), feed efficiency (1.19–1.26), percentage of dietary protein retained (45.8–47.5), gross energy utilization (46.5–49.4%), per cent survival (96.0–99.3) or terminal whole body and muscle proximate compositions. We conclude that SFM_EX can comprise ≥271 g kg^?1 of the dry diet or ≥22.7% of the digestible dietary protein of post‐smolt Atlantic salmon in seawater without any adverse effects on their performance.     

Chromosome localisation of genes for resistance to Heterodera schachtii, Cercospora beticola and Polymyxa betae using sets of Beta procumbens and B. patellaris derived monosomic additions in B. vulgaris

Beet cyst nematodes (BCN, Heterodera schachtii), Cercospora beticola, and rhizomania, caused by the beet necrotic yellow vein virus (BNYVV) and vectored by the soil-borne fungus Polymyxa betae, are the most serious diseases of sugar beet ( Beta vulgaris subsp. vulgaris). The wild Beta species of section Procumbentes are known to be completely resistant to H. schachtii, C. beticola and P. betae. Alien monosomic additions (2n=19), plants of cultivated beet (2n=18) carrying different individual chromosomes of B. procumbens (2n=18) or B. patellaris (2n=36), were tested in greenhouse experiments for resistance to these pathogens. Gene(s) conferring full resistance to the beet cyst nematode in B. patellaris are located on chromosome 1.1, and the other tested chromosomes of B. patellaris are not involved in the expression of resistance. Artificial inoculation under greenhouse conditions, with in vitro produced inoculum of C. beticola and spot-percentage rating of the disease intensity, showed that the high level of resistance that was observed in the wild species B. procumbens and B. patellaris was not found in any of the monosomic additions tested. It was suggested that genes on various chromosomes of the wild species are needed to express full resistance, and that the chromosomes of group 7 of B. patellaris and chromosome 7 of B. procumbens have the largest effect. The greenhouse tests for resistance to P. betae in B. patellaris derived monosomic additions showed that the addition families of group 4.1 have a strong partial resistance, while the addition families of group 8.1 appeared to be completely resistant to the pathogen. Resistance to P. betae in the two wild species as well as in the two resistant addition types did not exclude infection with BNYVV, but resulted in a considerable reduction of the virus concentration. It was concluded that resistance to the vector would complement virus resistance, and may provide a more effective and durable control of rhizomania. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sub-arm location of prolamin and EST-SSR loci on chromosome 1H<Superscript>ch</Superscript> from <Emphasis Type="Italic">Hordeum chilense</Emphasis>

Hordeum chilense Roem. et Schult. is a diploid wild South American barley that contains genes of interest for cereal breeding, many of them located on chromosome 1H^ch. In the current study, two H. chilense-wheat addition lines with deletions in the 1H^ch chromosome were used for sub-arm localization of five prolamin (glutenin and gliadin) loci and 33 EST-SSR marker loci on chromosome 1H^ch. The two sets of markers were distributed across five sub-arm chromosome regions. Three glutenin loci (Glu-H ^ch 2, Glu-H ^ch 3, Glu-H ^ch 4) together with the gliadin locus Gli-H ^ch 1 were located on the distal 20% of the 1H^chS arm, whereas the glutenin locus Glu-H ^ch 1 was on the proximal 88% region of 1H^chL. Among 33 EST-SSR marker loci, 7 (21.2%) were on the 1H^chS arm and, of them, 3 (9.1%) were on the distal 20% end and 4 (12.1%) on the proximal 80% region. The 26 loci (78.8%) on 1H^chL were distributed across three different regions: 18 (78.8%) in the proximal 88%, 3 (9.1%) in the distal 12% and 5 (15.2%) in a region less than 12% from the distal end. The deletions in the 1H^ch chromosome added to the common wheat background were thus shown to be useful for determining the sub-arm location of EST-SSR and prolamin loci. This could facilitate the identification of molecular markers linked to genes of agronomic interest and the isolation of such genes for use in common wheat improvement.