A variance-covariance structure to take into account repeated measurements and heteroscedasticity in growth modeling

This study proposes a within-subject variance-covariance (VC) structure to take into account repeated measurements and heteroscedasticity in a context of growth modeling. The VC structure integrates a variance function and a continuous autoregressive covariance structure. It was tested on a nonlinear growth model parameterized with data from permanent sample plots. Using a stand-level approach, basal area growth was independently modeled for red spruce (Picea rubens Sarg.) and balsam fir [Abies balsamea (L.) Mill.] in mixed stands. For both species, the implementation of the VC structure significantly improved the maximum likelihood of the model. In both cases, it efficiently accounted for heteroscedasticity and autocorrelation, since the normalized residuals no longer exhibited departures from the assumptions of independent error terms with homogeneous variances. Moreover, compared with traditional nonlinear least squares (NLS) models, models parameterized with this VC structure may generate more accurate predictions when prior information is available. This case study demonstrates that the implementation of a VC structure may provide parameter estimates that are consistent with asymptotically unbiased variances in a context of nonlinear growth modeling using a stand-level approach. Since the variances are no longer biased, the hypothesis tests performed on the estimates are valid when the number of observations is large.     

Multimycotoxin UPLC-MS/MS for tea, herbal infusions and the derived drinkable products

In recent years the consumption of tea and herbal infusions has increased. These hot drinks are consumed as daily drinks as well as for medicinal purposes. All tea varieties (white, yellow, green, oolong, black and puerh) originate from the leaves of the tea plant, Camellia sinensis. All extracts made of plant or herbal materials which do not contain Camellia sinensis are referred as herbal infusions or tisanes. During processing and manufacturing fungal contamination of the plant materials is possible, enabling contamination of these products with mycotoxins. In this study a multimycotoxin UPLC-MS/MS method was developed and validated for the analysis of the raw tea and herbal infusion materials as well as for their drinkable products. The samples were analyzed by ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), with a mobile phase consisting of variable mixtures of water and methanol with 0.3% formic acid. The limits of detection for the different mycotoxins varied between 2.1 μg/kg and 121 μg/kg for raw materials and between 0.4 μg/L and 46 μg/L for drinkable products. Afterward 91 different tea and herbal infusion samples were analyzed. Only in one sample, Ceylon melange, 76 μg/kg fumonisin B(1) was detected. No mycotoxins were detected in the drinkable products.     

Efficiency of SNP and SSR-based analysis of genetic diversity,population structure,and relationships among cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis> (L.) Walp.) germplasm from East Africa and IITA inbred lines

The extent of genetic diversity and relatedness of cowpea germplasm from East Africa are poorly understood. A set of 13 microsatellites (SSR) and 151 single nucleotide polymorphisms (SNPs) markers were applied to assess the levels of genetic diversity in a sample of 95 accessions of local cowpea germplasm and inbred lines of Vigna unguiculata. The average genetic diversity (D), as quantified by the expected heterozygosity, was higher for SSR loci (0.52) than for SNPs (0.34). The polymorphic information content was 0.48 for SSR and 0.28 for SNP while the fixation index was 0.095 for SSR and 0.15 for SNPs showing moderate differentiation and high gene flow among cowpea accessions from East African countries. The results of data analysis of both SSR and SNP markers showed similar clustering patterns suggesting a substantial degree of association between origin and genotype. Principal coordinate analysis (PCoA) with SSR and SNP markers showed that accessions were grouped into two and three broad groups across the first two axes, respectively. Our study found that SNP markers were more effective than SSR in determining the genetic relationship among East African local cowpea accessions and IITA inbred lines. Based on this analysis, five local cowpea accessions Tvu-13490, Tvu-6378, Tvu-13448, Tvu-16073, and 2305675 were identified to be tightly clustered sharing several common alleles with the drought tolerant variety Danila when analyzed with SSR and SNP markers. The findings will assist and contribute to future genetic diversity studies aimed at the genetic improvement of local Eastern Africa cowpea accessions for improved overall agronomic performance in general and breeding for drought tolerant in particular.     

Shading effects on canopy and tillering characteristics of continuously stocked palisadegrass in a silvopastoral system in the Amazon biome

In silvopastoral (SP) systems, forage responses depend on the microenvironment in which the plants develop. Our objective was to evaluate canopy and tillering characteristics of shaded 'Marandu' palisadegrass [Brachiaria brizantha (Hochst A Rich) Stapf, syn. Urochloa brizantha] under continuous stocking in a SP system. Treatments were one full sun (FS) and three shaded systems (silvopasture, SP) corresponding to distances from tree groves: 7.5 m north (SP1), and 15 m (SP2) and 7.5 m south (SP3) studied during two rainy seasons (Year 1 and Year 2). The tree in the SP system was Eucalyptus urograndis (hybrid of Eucalyptus grandis W. Hill ex Maiden × Eucalyptus urophylla S. T. Blake). The photosynthetic active radiation was greater in FS (923 μmol m^-2 s^-1), followed by SP2 (811 μmol m^-2 s^-1), SP1 (727 μmol m^-2 s^-1) and SP3 (673 μmol m^-2 s^-1). Forage accumulation in FS was 15% greater than the mean of SP1, SP2 and SP3 (10,663 kg DM/ha). There was no difference in net accumulation of leaf, stem and dead material, averaging 3,302, 3,420 and 4,063 kg DM/ha respectively. Leaf accumulation and accumulation rate were greater in Year 2, and leaf accumulation rate was similar among treatments (19 kg DM ha⁻¹ day⁻¹). Leaf proportion increased 14% from Year 1 to Year 2. Specific leaf area was greater for treatments SP1 and SP3 (193 cm²/g). Tiller population density was similar across treatments in Year 1. Shaded palisadegrass maintains leaf productivity similar to FS under continuous stocking in an SP system.     

Within-stem variations in mechanical properties of <Emphasis Type="Italic">Melia azedarach</Emphasis> planted in northern Vietnam

Within-stem variations in the mechanical properties of 17–19-year-old Melia azedarach planted in two sites in northern Vietnam were examined by destructive and nondestructive methods. Wood samples were collected from 10, 50, and 90% of the radial length from pith on both sides (North and South) at 0.3, 1.3, 3.3, 5.3, and 7.3 m heights above the ground. The mean values in whole trees of wood density (WD), modulus of rupture (MOR), modulus of elasticity (MOE), and dynamic modulus of elasticity (E_d) at 12% moisture content were 0.51 g/cm³, 78.58 MPa, 9.26 GPa, and 10.93 GPa, respectively. Within the stem, the radial position was a highly (p?<?0.001) significant source of variation in mechanical properties. MOR, MOE, and E_d increased from pith to bark. WD had a strong positive linear relationship with both MOR (r?=?0.85, p?<?0.001) and MOE (r?=?0.73, p?<?0.001). This suggests that it is potentially possible to improve mechanical properties through controlling WD. MOR had also a strong linear relationship with E_d (r?=?0.84, p?<?0.001). This indicates that E_d is a good indicator to predicting the strength of wood if the density of measured element is known. Besides, the stress wave method used in this study provides relatively accurate information for determining the stiffness of Melia azedarach planted in northern Vietnam.     

Determination of polychlorinated biphenyls by GC/MS with ultrasound-assisted extraction from shellfish

An efficient extraction method by ultrasound-assisted extraction (UAE) was developed for the extraction of polychlorinated biphenyls (PCB 28, 52, 101, 118, 130, 153, 180) from various shellfish. Analytes were determined by gas chromatography-mass spectrometry in selected ion monitoring (SIM) mode. All the samples were extracted by n-hexane and decontaminated by concentrated sulfuric acid. The resulting samples were determined by quantitative detection by comparing with external standards. The average recoveries of seven kinds of PCBs ranged from 80.92% to 93.89%, and the lowest detectable limit was 0.08-0.03 μg·kg⁻¹. The average concentrations of total PCBs were 14.12–30.61 μg·kg⁻¹ in the samples. This method was highly effective in reducing the cost and time for the pretreatment of samples. This method may be applied for the screening and monitoring of shellfish for organic pollutants in coastal waters.     

Culture system and long-term storage of culture media in the in vitro production of bovine embryos

The optimum culture system for in vitro matured and fertilised oocytes still remains to be clarified. Culture media (CM) for mammalian embryos are routinely prepared fresh for use and preserved under refrigeration during one or two weeks. The purposes of this work were (1) to compare the efficiency of a synthetic oviduct fluid (SOF) with two different bovine serum albumin (BSA) concentrations (3 and 8 g/L) for the in vitro production of bovine blastocysts, (2) to test the effect of timing on adding fetal calf serum (FCS) to the SOF, and (3) to evaluate the effects on bovine embryo development of freezing and lyophilisation as procedures for preserving the SOF. Supplementation of SOF with 3 g/L BSA increased Day-7 blastocyst expansion rates (18.3 ± 1.6 vs. 14.4 ± 0.7; P < 0.05), although no differences in hatching rates were found. Addition of FCS to SOFaa (SOF with amino acids) medium supplemented with sodium citrate (SOFaaci) at 48 and at 72 h post-insemination (PI) allowed obtaining higher Day-6 embryo development rates than when FCS was added at 18 or 96 h PI (Day-6 morulae + blastocyst rate: 30.0 ± 1.1, 40.8 ± 1.1, 43.9 ± 2.3 and 39.3 ± 0.5 for FCS addition at 18, 48, 72 and 96 h, respectively). Hatching rates were significantly improved when serum was added at 72 h PI. Finally, both refrigeration and lyophilisation appeared as useful cryopreservation procedures for SOFaaci, although a significant loss of its ability to support embryo development, compared to the control fresh culture medium, was observed.     

Application of ELISA-based kit for detecting AOZ and determining its clearance in eel tissues

Furazolidone, an antibacterial drug that was once widely used in the livestock industry and aquaculture, is now prohibited in numerous countries. It is difficult to detect residual furazolidone because it is readily metabolized in animal tissues but, by using and liquid chromatography coupled with tandem mass spectrometry, its metabolite, 3-amino-2-oxazolidinone (AOZ) can be detected. Here we describe the validity of an enzyme-linked immunosorbent assay (ELISA) kit to detect AOZ in Japanese eel Anguilla japonica tissue. ELISA is capable of detecting AOZ at 1.0 μg/kg in an eel sample with excellent accuracy and precision. Our results show that ELISA is suitable for regulatory purposes and for studying the fate of AOZ residues in eel treated with furazolidone. To measure the persistence of AOZ in eel tissues, eels (1.4–6.5g) were immersed in tanks containing 2 and 10 mg furazolidone/L for 3 h, and then maintained in a tank supplying well water for the next 160 days. The half-lives of AOZ, calculated from the linear terminal part of the excretion curve, were 25.0 days in muscle and 21.6 days in liver from fish exposed to 2 mg/L furazolidone. In the eels treated with 10 mg/L furazolidone, by contrast, high levels of AOZ were detected in liver and muscle, but the half-lives of AOZ were similar to those in fish treated with 2 mg/L furazolidone. The half-lives of AOZ in eel tissues were prolonged by the condition of low water temperature.     

Mechanical insights into the effect of fluctuation in soil moisture on nitrous oxide emissions from paddy soil

Paddy fields are subjected to fluctuating water regimes as a result of the alternate drying and wetting water management, which often incurs a sensitive change in N₂O emissions from paddy soils. However, how the soil moisture regulates the emission of N₂O from paddy soil remains uncertain. In this study, three incubation experiments were designed to study the effects of constant and fluctuating soil moisture on N₂O emission and the sources of N₂O emission from paddy soil. Results showed that the N₂O emission from paddy soil at 100 % WHC (water-holding capacity) was higher than that at 40, 65, 80, 120, and 160 % WHC, indicating that 100 % WHC was the optimum soil moisture content for N₂O emission under the incubation experiment. Small peak of N₂O flux appeared when the soil moisture content from 250 % WHC decreased near to 100 % WHC, lower than that triggered by nitrogen (N) fertilization, which was mainly owing to the low NH₄ ⁺ concentration at this period. Nitrification dominated the emissions of N₂O from paddy soil at 250 % WHC (54.96 %), higher than that of nitrification-coupled denitrification (6.74 %) and denitrification (38.3 %). The contribution of denitrification to N₂O emissions (44.10 %) was equivalent to that of nitrification (44.45 %) in soil at 100 % WHC, which was higher than that of 250 % WHC treatment. In conclusion, the finding suggested that the peak of N₂O in paddy soils during midseason aeration could be attributed to the occurrence of optimum soil moisture under sufficient N availability, favorable for the production and accumulation of N₂O.     

Mucosal and systemic isotype-specific antibody responses and protection in conventional pigs exposed to virulent or attenuated porcine epidemic diarrhoea virus

Eleven-day-old conventionally reared piglets were inoculated orally with two different doses of the cell-culture adapted strain CV-777 of the porcine epidemic diarrhoea virus (PEDV) or the virulent isolate of the same strain and challenged with the same virulent PEDV 3 weeks later. Pigs inoculated with the two doses of the attenuated virus did not show any typical sign of the disease, and virus shedding was not frequent. In contrast, 31% of pigs exposed to the virulent PEDV developed diarrhoea and virus shedding was demonstrated in 100%. At different postinoculation day (PID) and postchallenge day (PCD) virus-specific antibody-secreting cells (ASC) in gut associated lymphoid tissues (duodenum and ileum lamina propria and mesenteric lymph nodes) and systemic locations (blood and spleen) were assessed by enzyme-linked immunospot (ELISPOT). Only a small response was detected in the groups inoculated with attenuated PEDV, whereas in the group previously exposed to the virulent virus on PID 21 a large number of IgG and IgA ASC was detected. Isotype-specific antibody responses in serum were investigated by ELISA. IgG responses were detected in all groups, although the highest response corresponded to the group inoculated with virulent virus and only this group showed an IgA response. The pigs exposed to virulent PEDV were completely protected against the challenge with a higher dose of the same virulent virus on PID 21 and none of them shed the virus. The pigs inoculated with the attenuated strain were partially protected against the challenge, and 25% of the low dose- and 50% of the high dose-exposed pigs did not shed virus after challenge. All the pigs from a control group, not previously exposed to the virus, excreted the virus in faeces. A strong positive correlation was established between protection and the ASC responses detected in gut associated lymphoid tissues and blood at the challenge day and also between protection and serum isotype-specific antibody titers on that day. In addition, the IgA and IgG ASC responses detected in the blood on PID 21 also correlated with the responses found in the gut associated lymphoid tissues. The ASC and serum antibody responses after the challenge corresponded to a secondary immune response in the groups inoculated with attenuated virus, whereas a primary response was evident in the control group. No increase was seen in any of the parameters studied in the pigs inoculated with virulent PEDV.