The influence of narrow-leafed lupin seed fermentation on their chemical composition and ileal digestibility and microbiota in growing pigs

The aims of this study were to provide a controlled fermentation process of blue lupin seeds (Lupinus angustifolius, cv. Neptun), monitor the changes in seed composition and determine the influence of the fermentation on the apparent ileal digestibility (AID) of crude protein (CP) and amino acids in growing pigs, compared with raw lupin seeds. The fermentation with bacteria and yeast was conducted for 24 h at 25ºC under aerobic conditions. Seed fermentation increased the contents of CP, fibre, fat and ash and most of the analysed amino acids but reduced the levels of the nitrogen-free extractives. Furthermore, fermentation decreased the contents of raffinose family oligosaccharides and phytic acids but increased the alkaloid content. The AID was estimated on three barrows (mean initial body weight 25 kg), surgically fitted with a T-cannula in the distal ileum. The pigs received three diets, each for 6 d, within three experimental periods (3 × 3 Latin Square design). The diets contained soybean meal (Group SBM), raw lupin seeds (Group RL) or fermented lupin seeds (Group FL) as solely protein sources. Fermentation had a positive impact on the AID of CP and methionine, cysteine, isoleucine, leucine, phenylalanine and valine (p < 0.05). Feeding raw or fermented lupin seeds did not affect the microbial status of the ileal digesta. Moreover, ammonia content in the caecal digesta did not differ between Groups RL and FL, although it was significantly higher than in Group SBM. It can be concluded that the fermentation process modified the chemical composition of nutrients in seeds, which can influence the digestibility and utilisation of the fermentation product in animal diets compared to raw seeds.     

Relationships between nitrogen,amino acids and storage proteins in Lupinus albus seeds

Lupinus albus seeds (20 samples from 10 different lines or cultivars) with protein contents distributed from 23.8 to 48.4% were analysed for their amino acid composition with great accuracy (from 6 hydrolysates per sample). Amino acid levels in seeds increased as linear functions of nitrogen content with correlation coefficients close to unit in all the lupin genotypes and phenotypes. Hence the composition of any lupin seed sample can be predicted from its nitrogen content. Amino acid in total seed protein varied as hyperbolic functions of nitrogen content. The same was true for nitrogen-to-protein conversion factors. By controst, the composition of storage proteins accumulated in seeds remained constant and independent of seed protein content.     

Identification of a Low Digestibility δ-Conglutin in Yellow Lupin (Lupinus luteus L.) Seed Meal for Atlantic Salmon (Salmo salar L.) by Coupling 2D-PAGE and Mass Spectrometry

The need of quality protein in the aquaculture sector has forced the incorporation of alternative plant proteins into feeding diets. However, most plant proteins show lower digestibility levels than fish meal proteins, especially in carnivorous fishes. Manipulation of protein content by plant breeding can improve the digestibility rate of plant proteins in fish, but the identification of low digestibility proteins is essential. A reduction of low digestibility proteins will not only increase feed efficiency, but also reduce water pollution. Little is known about specific digestible protein profiles and/or molecular identification of more bioavailable plant proteins in fish diets. In this study, we identified low digestibility L. luteus seed proteins using Atlantic salmon (Salmo salar) crude digestive enzymes in an in vitro assay. Low digestibility proteins were identified by comparing SDS-PAGE banding profiles of digested and non-digested lupin seed proteins. Gel image analysis detected a major 12 kDa protein band in both lupin meal and protein isolate digested products. The 12 kDa was confirmed by 2D-PAGE gels and the extracted protein was analyzed with an ion trap mass spectrometer in tandem mass mode. The MS/MS data showed that the 12 kDa low digestibility protein was a large chain δconglutin, a common seed storage protein of yellow lupin. Comparison of the protein band profiles between lupin meal and protein isolates showed that the isolatation process did not affect the low digestibility of the 12 kDa protein.     

Nutritional evaluation of fermented black gram (Phaseolus mungo) seed meal in compound diets for rohu, Labeo rohita (Hamilton), fingerlings

Six isonitrogenous (approximately 35% crude protein) and isocaloric (approximately 4.0 kcal g⁻¹) diets were formulated incorporating raw and fermented black gram, Phaseolus mungo, seed meal at 20%, 30% and 40% levels by weight into a fishmeal‐based control diet fed to rohu, Labeo rohita, fingerlings (mean weight, 1.81 ± 0.21 g) for 80 days for a study of fish performance. A particular bacterial strain (Bacillus sp.) isolated from the intestine of adult common carp (Cyprinus carpio) reared in the wild having significant amylolytic, cellulolytic, lipolytic and proteolytic activities was used for fermentation of seed meal for 15 days at 37 ± 2°C. Fermentation of P. mungo seed meal was effective in significantly reducing the crude fibre content and antinutritional factors such as tannins and phytic acid, and enhancing available free amino acids and fatty acids. In terms of growth, feed conversion ratio and protein efficiency ratio, the 30% fermented black gram seed meal incorporated diet resulted in a significantly (P < 0.05) better performance of rohu fingerlings. In general, growth and feed utilization efficiencies of diets containing fermented seed meal were superior to diets containing raw seed meal. The apparent protein digestibility (APD) values decreased with increasing levels of raw seed meal in the diets. The APD for raw seed meal was lower at all levels of inclusion in comparison to those for the fermented seed meals. The maximum deposition of protein in the carcass was recorded in fish fed the diet containing 40% fermented seed meal. The results indicate that fermented black gram seed meal can be incorporated in carp diets up to the 30% level compared to the 10% level of raw seed meal.     

Improvement of nutritive value of grass pea (Lathyrus sativus) seed meal in the formulated diets for rohu, Labeo rohita (Hamilton) fingerlings after fermentation with a fish gut bacterium

Eight isonitrogenous (35% crude protein approximately) and isocaloric (4.0 kcalg(-1) approximately) diets were formulated incorporating raw and fermented grass pea (Lathyrus sativus) seed meal at 10%, 20%, 30% and 40% levels by weight into a fish meal based diet and fed to rohu, Labeo rohita, fingerlings for 80 days and fish performance was studied. A particular bacterial strain (Bacillus sp.) isolated from the intestine of adult common carp (Cyprinus carpio) reared in the wild having significant amylolytic, cellulolytic, lipolytic and proteolytic activities were used for fermentation of seed meal for 15 days at 37 degrees C. Fermentation of grass pea seed meal was effective in significantly reducing the crude fibre content and anti-nutritional factors, such as tannins, phytic acid and the neurotoxin, beta-ODAP and enhancing the available free amino acids and fatty acids. In terms of growth response, feed conversion ratio and protein efficiency ratio, 30% fermented grass pea seed meal incorporated diet resulted in significantly (P < 0.05) better performance of rohu fingerlings. In general, growth and feed utilization efficiencies of fish fed diets containing fermented seed meal were superior to those fed diets containing raw seed meal. The apparent protein digestibility (APD) values decreased with increasing levels of raw seed meal in the diets. The APD for raw seed meal was lower at all levels of inclusion in comparison to those for the fermented seed meals. The highest deposition of carcass protein was recorded in fish fed the diet containing 40% fermented seed meal. The results indicated that fermented grass pea seed meal can be incorporated in carp diets up to 30% level compared to 10% level of raw seed meal.     

Identification of chromosome regions controlling seed storage proteins of narrow-leafed lupin (Lupinus angustifolius)

Narrow-leafed lupin (Lupinus angustifolius L.) is a valuable legume crop for animal feed and human health food because of its high proteins content. However, the genetics of seed storage proteins is unclear, limiting further improvement of protein quantity and quality. In this study, matrix-assisted laser desorption/ionization time of flight mass spectrometry was used for the first time to analyze lupin seed storage proteins and the spectra generated was treated as markers to investigate the chromosome locations controlling seed storage proteins in the narrow-leafed lupin. In a recombinant inbred line population of 89 individuals, 48 polymorphic protein peaks were identified and seven of which were successfully mapped onto four existing linkage groups: two on NLL-04, three on NLL-05, one on NLL-07 and one on NLL-14, with LOD values ranging from 2.6 to 7.7 confirming a significant linkage. Most protein-based markers showed distorted segregation and were failed to be integrated into the reference map. Among them, 31 were grouped into six clusters and the other ten were totally unlinked. This study provides a significant clue to study the comparative genomics/proteomics among legumes as well as for protein marker-assisted breeding. The distribution pattern of genes controlling seed storage protein revealed in this study probably exists universally among legumes or even all plants and animals. Whether genes controlling seed storage protein share the same gene expression pattern controlling other enzymes and what is the mechanism behind it are the questions which remain to be answered in the future.     

Molecular studies on the ecology of Listeria monocytogenes in the smoked fish processing industry

We have applied molecular approaches, including PCR-based detection strategies and DNA fingerprinting methods, to study the ecology of Listeria monocytogenes in food processing environments. A total of 531 samples, including raw fish, fish during the cold-smoking process, finished product, and environmental samples, were collected from three smoked fish processing facilities during five visits to each facility. A total of 95 (17.9%) of the samples tested positive for L. monocytogenes using a commercial PCR system (BAX for Screening/Listeria monocytogenes), including 57 (27.7%) environmental samples (n = 206), 8 (7.8%) raw material samples (n = 102), 23 (18.1%) samples from fish in various stages of processing(n = 127), and 7 (7.3%) finished product samples (n = 96). L. monocytogenes was isolated from 85 samples (16.0%) using culture methods. Used in conjunction with a 48-h enrichment in Listeria Enrichment Broth, the PCR system had a sensitivity of 91.8% and a specificity of 96.2%. To track the origin and spread of L. monocytogenes, isolates were fingerprinted by automated ribotyping. Fifteen different ribotypes were identified among 85 isolates tested. Ribotyping data established possible contamination patterns, implicating raw materials and the processing environment as potential sources of finished product contamination. Analysis of the distribution of ribotypes revealed that each processing facility had a unique contamination pattern and that specific ribotypes persisted in the environments of two facilities over time (P < or = 0.0006). We conclude that application of molecular approaches can provide critical information on the ecology of different L. monocytogenes strains in food processing environments. This information can be used to develop practical recommendations for improved control of this important food-borne pathogen in the food industry.     

Changes in water status and water distribution in maturing lupin seeds studied by MR imaging and NMR spectroscopy

The changes in water distribution in maturing lupin (Lupinus luteus L.) seeds were visualized with magnetic resonance imaging (MRI). MRI data showed local inhomogeneities of water distribution inside the seed. At the late seed-filling stage the most intense signal was detected in the seed coat and the outer parts of cotyledons in the hilum area, but during maturation drying the decline in MR image intensity was faster in the outer part of the seed than in the central part. The changes in water status were characterized by NMR spectroscopy. Analyses of T(2) relaxation times revealed a three-component water proton system in maturing lupin seeds. Three populations of protons found during seed maturation, each with a different magnetic environment causing a different relaxation rate, were correlated with three fractions of water (structural, intracellular, and extracellular) that were observed during seed germination. This study provides evidence that lupin seeds have similar states of the different water components with regard to seed moisture content at two distinct physiological stages, seed maturation and germination. The unique feature of maturing lupin seeds is the presence of the high (1)H-NMR signal in areas corresponding to the vascular bundles. Tissue localization of dehydrins showed the presence of dehydrin protein in the area of vascular tissue. An anti-dehydrin antibody detected three polypeptides in lupin embryos with molecular masses of 73, 43 and 28 kDa, respectively. The temporal pattern of dehydrin protein accumulation correlates well with seed desiccation.     

Combined 2D electrophoretic approaches for the study of white lupin mature seed storage proteome

Seed proteome analysis by 2D IEF/SDS-PAGE techniques is challenging for the intrinsic difficulties related to quantitative disparity of the seed proteins, i.e. storage and non-storage proteins, their polymorphic nature, the extensive post-translational modifications and the paucity of deposited primary structures available. Conversely, 2D maps of seed proteomes can be extremely useful for a number of fundamental and applied investigations. In this work, we have used a combination of two experimental approaches to identify the main protein components of an emerging protein-rich legume seed, that is white lupin seed (Lupinus albus, L.). One is the canonical proteomic approach including 2D electrophoretic separation and mass spectrometry of selected trypsin-digested polypeptides; the other approach is a group comparative 2D electrophoretic analysis of cotyledonary protein families. To this second purpose, the three main families of lupin seed proteins, namely alpha-conglutins, the 11S globulin fraction, beta-conglutins, the 7S globulin fraction, and gamma-conglutin, a basic 7S protein, were isolated by conventional biochemical techniques and their 2D reference maps were compared with the total protein map. With the first approach 37 out of 40 spots, making up about 35% of total spot volumes in the 2D map, were found to belong to the main seed protein families. Thanks to cDNA-deduced lupin storage protein sequences, determined on purpose and deposited, most of the identification statistical parameters were very good. Moreover, it was possible to identify several endogenously proteolysed subunits in the map. The second comparative approach, beside confirming these attributions, allowed to allocate 124 polypeptides within the three main lupin protein families. These two approaches proved to be mutually validating and their combined use was effective for the establishment of a seed proteome map even in the case of sequence and protein post-translational processing lack of information. The results obtained also extend our knowledge of the seed storage protein polymorphism of white lupin.     

Effects of dehulling,steam-cooking and microwave-irradiation on digestive value of white lupin (Lupinus albus) seed meal for rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)

A digestibility trial was conducted to assess the effect of dehulling, steam-cooking and microwave-irradiation on the apparent digestibility of nutrients in white lupin (Lupinus albus) seed meal when fed to rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). Six ingredients, whole lupin seed meal (LSM), dehulled LSM, dehulled LSM steam-cooked for 15 or 45 min (SC15 and SC45, respectively) and LSM microwave-irradiated at 375 or 750 W (MW375 and MW750, respectively), were evaluated for digestibility of dry matter, crude protein (CP), lipids, nitrogen-free extractives (NFE) and gross energy (GE). The diet-substitution approach was used (70% reference diet + 30% test ingredient). Faeces from each tank were collected using a settlement column. Dehulled LSM showed higher levels of proximate components (except for NFE and crude fibre), GE and phosphorus in comparison to whole LSM. Furthermore, SC15, SC45, MW375 and MW750 showed slight variations of chemical composition in comparison to dehulled LSM. Results from the digestibility trial indicated that dehulled LSM, SC15, SC45 and MW375 are suitable processing methods for the improvement of nutrients’ apparent digestibility coefficient (ADC) in whole LSM. MW750 showed a lower ADC of nutrients (except for CP and lipids for rainbow trout) in comparison with MW350 for rainbow trout and Atlantic salmon, suggesting a heat damage of the ingredient when microwave-irradiation exceeded 350 W.     

Regulation of storage lipid metabolism in developing and germinating lupin (<Emphasis Type="Italic">Lupinus</Emphasis> spp.) seeds

The main storage compound in lupin seeds is protein, whose content can reach up to 45–50 % of dry matter. However, seeds of some lupin species can also contain quite a large amount of storage lipid. The range of lipid content in lupin seeds is from about 6 to about 20 % of dry matter. Storage lipid in developing seeds is synthesized mainly from sugars delivered by mother plants. During seed germination, one of the main end-products of storage lipid breakdown is also sugars. Thus, the sugar level in tissues is considered an important regulatory agent, during both lipid accumulation and lipid breakdown. Generally, in developing legume seeds, there is a strong negative relation between accumulation of storage protein and storage lipid. Results obtained in developing lupin cotyledons cultured in vitro pointed to the possibility of a positive relation between protein and lipid accumulation. Such a positive effect could be caused by nitrate. During lupin seed germination and seedling development, the utilization of storage lipid is enhanced under sugar deficiency conditions in tissues and is controlled at the gene expression level. However, under sugar starvation conditions, autophagy is significantly enhanced, and it can cause disturbances in storage lipid breakdown. The hypothesis of pexophagy, i.e., autophagic degradation of peroxisomes under sugar starvation conditions during lupin seed germination, has been taken into consideration. The flow of lipid-derived carbon skeletons to amino acids was discovered in germinating lupin seeds, and this process is clearly more intense in sucrose-fed embryo axes. At least four alternative or mutually complementary pathways of carbon flow from storage lipid to amino acids in germinating lupin seeds are postulated. The different strategies of storage compound breakdown during lupin seed germination are also discussed.     

Variability for protein and oil quality in Lupinus albus

Amino acid composition and fatty acid composition were determined on seed samples of a range of white lupin (Lupinus albus) cultivars and accessions grown in either of two environments.Variability between genotypes was found for lysine, arginine and glutamic acid content, but not for the concentrations of other amino acids. The deficiency in sulphurcontaining amino acids, typical of legume proteins, was evident, with methionine and cyst(e)ine totalling only 2.2% of the protein. Variability was limited, indicating that improvement by breeding would be impracticable. Lupinus albus differed slightly from other lupin species in amino acid composition, having higher levels of threonine, tyrosine and isoleucine, but a lower level of glutamic acid than both L. angustifolius and L. luteus. Four low-alkaloid lines of L albus each had higher lysine content than the high-alkaloid line, but ‘Kiev Mutant’, despite earlier claims, had a lysine level no higher than the other three low-alkaloid lines.Fatty acid composition of the seed oil varied considerably between genotypes. Oleic acid ranged from 43.6 to 54.4% and linolenic acid from 6.7 to 15.2%, these two fatty acids being negatively correlated at one site. Linoleic acid content varied between 17.2 and 26.9% and was not correlated with other fatty acids. Total oil content averaged 9.6% with little variability between lines.It is concluded that, relative to other lupin species, L. albus has a more favourable amino acid profile for its utilisation in cereal-based diets for animals, particularly if the energy source is wheat, which is deficient in threonine. The higher oil content would be an important energy benefit to such diets and may allow their protein/energy balance to be maintained at higher levels of incorporation of L. albus seed meal than is possible with other lupin species.     

Seed-borne cucumber mosaic virus infection of narrow-leafed lupin (Lupinus angustifolius) in Western Australia

In 1986 in Western Australia, cucumber mosaic virus (CMV) infection was widespread in breeders' selections of narrow-leafed lupin (Lupinus angustifolius), and in collections of lupin cvs and wild L. angustifolius lines. When seed of some of these selections and cvs was sown, seed-borne CMV was detected in seedlings. Infection of F₁ progenies was traced to use of infected parent plants. CMV was also widespread in 25 seed crops of the new lupin cv. Wandoo but not in 42 seed crops of the new cv. Danja. When samples of the seed sown in 1986 were tested, CMV was detected in 3 - 34% of seedlings of cv. Wandoo but in none of cv. Danja. Following intensive roguing of symptom-bearing plants in the 1986 seed crop of new lupin cv. Gungurru, the level of seedling infection with CMV in seed samples after harvest was 0·1-0·2%. CMV was detected in 6 - 8%, 0·6-5% and 0 - 18% of seedlings from seed samples of established lupin cvs Chittick, Yandee and Illyarrie respectively. Highest levels of seed transmission were in seed from crops grown in high rainfall areas. When a sample of cv. Wandoo seed was graded for size by sieving, CMV was detected in seedlings grown from seed in all grades, but the smallest grade contained the highest level of infection. When seed was collected from pods at different positions on plants in a CMV-infected crop of cv. Illyarrie, seed from primary pods transmitted the virus to seedlings at a 3% rate, seed from first order lateral pods at 8% while seed from second and third order lateral pods transmitted at 13%. Examination of CMV-infected lupin crops indicated that seed-infected plants competed poorly and tended to be shaded out in dense crops but to survive in sparse crops. In 1987 during drought conditions after seeding, plant mortality was greater with seed-infected seedlings than with healthy seedlings despite wide plant spacing. An isolate of CMV from subterranean clover (Trifolium subterraneum) induced severer symptoms in lupins than four isolates from lupin; only the subterranean clover isolate prevented seed production. In tests at one lupin breeding site, CMV was found in 15 species of weeds and volunteer legumes. Fumaria officinalis, Stachys arvensis and volunteer lupins were most frequently infected.     

A quinolizidine alkaloid O-tigloyltransferase gene in wild and domesticated white lupin (Lupinus albus)

Wild white lupins have high levels of alkaloids, which cause a bitter taste, whereas domesticated white lupin varieties have a very low content of alkaloids in seeds. Genes for bitterness from wild white lupins are a contamination threat to domesticated white lupin via cross‐pollination. The gene(s) for alkaloid synthesis have not been clearly identified, and the associated molecular background among wild white lupin, domesticated and contaminated domesticated plant materials is unknown. So far, only tigloyl‐CoA:(?)‐13alpha‐hydroxymultiflorine/(+)‐13alpha‐hydroxylupanine O‐tigloyltransferase (HMT/HLTase) cDNA has been cloned based on protein analysis, which was suggested as encoding a quinolizidine alkaloid transferase regulating quinolizidine alkaloid biosynthesis. This gene has not yet been well characterised in important white lupin genotypes. In this study, we found that the majority of the intron sequence of the HMT/HLTase gene differed between wild white lupin accessions P25758 and P27593, and between the commercial varieties. The expression pattern as well as the expression level of the HMT/HLTase gene showed no difference between the P25758 and the low‐alkaloid variety Kiev mutant, suggesting the expression of the HMT/HLTase gene has no correlation with bitterness. However, the intron sequence is useful as a DNA marker in the identification of the contamination source of bitter seeds in commercial lupin seed lots.     

Limits to sulfur accumulation in transgenic lupin seeds expressing a foreign sulfur-rich protein

The low sulfur amino acid content of legume seeds restricts their nutritive value for animals. We have investigated the limitations to the accumulation of sulfur amino acids in the storage proteins of narrow leaf lupin (Lupinus angustifolius) seeds. Variation in sulfur supply to lupin plants affected the sulfur amino acid accumulation in the mature seed. However, when sulfur was in abundant supply, it accumulated to a large extent in oxidized form, rather than reduced form, in the seeds. At all but severely limiting sulfur supply, addition of a transgenic (Tg) sink for organic sulfur resulted in an increase in seed sulfur amino acid content. We hypothesize that demand, or sink strength for organic sulfur, which is itself responsive to environmental sulfur supply, was the first limit to the methionine (Met) and cysteine (Cys) content of wild-type lupin seed protein under most growing conditions. In Tg, soil-grown seeds expressing a foreign Met- and Cys-rich protein, decreased pools of free Met, free Cys, and glutathione indicated that the rate of synthesis of sulfur amino acids in the cotyledon had become limiting. Homeostatic mechanisms similar to those mediating the responses of plants to environmental sulfur stress resulted in an adjustment of endogenous protein composition in Tg seeds, even when grown at adequate sulfur supply. Uptake of sulfur by lupin cotyledons, as indicated by total seed sulfur at maturity, responded positively to increased sulfur supply, but not to increased demand in the Tg seeds.     

膏桐种子形态、粗脂肪含量与环境因子关系

对云南省两个不同地区膏桐（Jatropha curcas）种子形态特征,粗脂肪含量与环境因子关系开展研究,结果表明：湿热环境的景洪较干热环境的元谋膏桐种子成熟散布期提前,形态特征也优于元谋膏桐种子（P〈0．01）;土壤有机质是影响膏桐种子粗脂肪积累最重要的因子,对膏桐种子粗脂肪积累起显著正效应;干旱胁迫也是影响膏桐种子粗脂肪积累的一个重要的限制因子;景洪膏桐种子粗脂肪含量、含水量较元谋膏桐种子更高,但粗脂肪含量与含水量比率较低（P〈0．01）;具有较高粗脂肪含量的膏桐种子与其含水量的比率可能是植物对干旱胁迫适应的一种机制,其对物种的适应有着重要的生态学意义。     

Proteomic characterization of seeds from yellow lupin (Lupinus luteus L.)

Yellow lupin (Lupinus luteus L.) is a legume crop containing a large amount of protein in its seeds. In this study, we constructed a seed‐protein catalog to provide a foundation for further study of the seeds. A total of 736 proteins were identified in 341 2DE spots by nano‐LC‐MS/MS. Eight storage proteins were found as multiple spots in the 2DE gels. The 736 proteins correspond to 152 unique proteins as shown by UniRef50 clustering. Sixty‐seven of the 152 proteins were associated with KEGG‐defined pathways. Of the remaining proteins, 57 were classified according to a GO term. The functions of the remaining 28 proteins have yet to be determined. This is the first yellow lupin seed–protein catalog, and it contains considerably more data than previously reported for white lupin (L. albus L.).     

Influence of the grinding level and extrusion on the nutritional value of lupin seed (Lupinus albus) for cattle in the context of the Dutch protein evaluation system

The experiment was conducted on double-muscled Belgian Blue bulls, fitted with rumen, duodenal and ileal cannulas, according to a 5 × 5 Latin square design with one missing animal. All diets contained 320 g/kg of lupin seed (Lupinus albus, var. Lublanc) on a dry matter (DM) basis. Four grinding treatments of raw lupins (RAW) were used to obtain median particle sizes of approximately 0.5, 2.0, 4.2 or 6.0 mm. One extrusion treatment (EXTR) was also used and consisted of heating to 180 °C for 30 s. The pH and ammonia nitrogen (NH₃–N) concentration kinetics in the rumen liquid differed between RAW and EXTR. The latter induced larger variation in these parameters after the meal. Quadratic effects of the grinding level on rumen fermentation parameters were observed at several sampling times for RAW. The grinding level had also quadratic effects on the intestinal digestibility of DM, organic matter (OM) and N, reaching a maximum with treatment 4.2 mm. The treatment 0.5 mm tended to reduce the microbial N flow at the duodenum whereas EXTR tended to increase the non NH₃–N flow. The total digestible flow of N tended to increase with the intermediate grinding levels and EXTR. Lupin protein degradability was calculated at 0.78, 0.74, 0.65, 0.82 and 0.70 for the 0.5, 2.0, 4.2, 6.0 mm treatments and EXTR, respectively. Treatments 2.0 and 4.2 mm increased the digestible protein in the small intestine (DVE) content by more than 40% compared with treatment 0.5 mm. The balance between rumen fermentable N and energy (OEB) was reduced by 15% with the 4.2 mm and EXTR. Compared with treatment 4.2 mm, EXTR did not greatly improve the nutritional value of lupin seed. The results suggested that lupin seed should be coarsely ground or flattened to obtain a mean particle size between 2.0 and 4.2 mm for cattle feed, but that an insufficient grinding level (treatment 6.0 mm) induced a higher degradability of lupin protein, probably due to more intense rumination. Under our experimental conditions, the extrusion did not sufficiently improve the nutritional value of the seed to be economically viable. These results show that feeding standards should consider the influence of the grinding level of legume seeds in order to assess their nutritional value accurately.     

Childhood Diarrhea Exhibits Spatiotemporal Variation in Northwest Ethiopia: A SaTScan Spatial Statistical Analysis

Background

Childhood diarrhea continues to be a public health problem in developing countries, including Ethiopia. Detecting clusters and trends of childhood diarrhea is important to designing effective interventions. Therefore, this study aimed to investigate spatiotemporal clustering and seasonal variability of childhood diarrhea in northwest Ethiopia.

Methods

Retrospective record review of childhood diarrhea was conducted using quarterly reported data to the district health office for the seven years period beginning July 1, 2007. Thirty three districts were included and geo-coded in this study. Spatial, temporal and space-time scan spatial statistics were employed to identify clusters of childhood diarrhea. Smoothing using a moving average was applied to visualize the trends and seasonal pattern of childhood diarrhea. Statistical analyses were performed using Excel^® and SaTScan programs. The maps were plotted using ArcGIS 10.0.

Results

Childhood diarrhea in northwest Ethiopia exhibits statistical evidence of spatial, temporal, and spatiotemporal clustering, with seasonal patterns and decreasing temporal trends observed in the study area. A most likely purely spatial cluster was found in the East Gojjam administrative zone of Gozamin district (LLR = 7123.89, p <0.001). The most likely spatiotemporal cluster was detected in all districts of East Gojjam zone and a few districts of the West Gojjam zone (LLR = 24929.90, p<0.001), appearing from July 1, 2009 to June 30, 2011. One high risk period from July 1, 2008 to June 30, 2010 (LLR = 9655.86, p = 0.001) was observed in all districts. Peak childhood diarrhea cases showed a seasonal trend, occurring more frequently from January to March and April to June.

Conclusion

Childhood diarrhea did not occur at random. It has spatiotemporal variation and seasonal patterns with a decreasing temporal trend. Accounting for the spatiotemporal variation identified in the study areas is advised for the prevention and control of diarrhea.     

Varying phosphorus supply and development,growth and seed yield in narrow-leafed lupin

Narrow-leafed lupin (Lupinus angustifolius L.) is usually grown in sandy, acidic and phosphorus (P) deficient soil with low yield and variable harvest index. This study aimed to examine the effects of varying P supply on lupin growth, seed yield and harvest index. Non-abscission plants (cv. Danja) were grown in Lancelin sand at seven rates of P supply (5, 10, 15, 20, 25, 30 or 40 mg kg^–1) in a naturally-lit glasshouse. The rate of leaf emergence, flowering time and flower number were decreased or delayed by low P supply (5, 10 or 15 mg kg^–1), with no differences at P rates higher than 20 mg kg^–1. High P supply (25, 30 or 40 mg kg^–1) increased plant seed yield and harvest index largely by increasing the number of pods and consequently yield on the lateral branches, but had less effect on the number of seeds per pod and seed size. Seed yield and seed P concentration continued to increase up to 40 mg P kg^–1but harvest index plateaued at 25 mg P kg^–1, indicating that low P supply decreased reproductive growth more than vegetative growth in narrow-leafed lupin.