Subcellular localization and isoenzyme pattern of peroxidase and polyphenol oxidase in beet root (Beta vulgaris L.)

The two enzymes involved in enzymatic browning reactions, polyphenol oxidase (PPO) and peroxidase (PO), have been partially purified and extracted from different fractions of beet root. PPO is mainly located in the membrane fraction, and it was also found in the soluble fraction. In both cases PPO was in its latent state. However, PO activity was higher in the soluble fraction than in the membrane fraction. Nevertheless, the highest values of specific activity for PO were obtained from the solubilized enzyme from acetone powders. Under native isoelectric focusing (IEF), several PPO isoenzymes were present in the pH range of 4.8-5.8. All of these isoenzymes shared a single band with a similar apparent mass under sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PO was also analyzed by IEF, showing a complex isoenzyme pattern in all fractions. The characteristic basic PO isoenzyme of high pI found in both the soluble fraction and the solubilized enzyme from acetone powders was not detected in the membrane fraction. The kinetic characterization of PPO and PO from all fractions was carried out.     

Variation and genetic diversity for gliadins in Spanish spelt wheat accessions

Gliadin composition has been analysed in 403 accessions of spelt wheat (Triticum aestivum ssp. spelta); 61 different patterns were found for the -gliadins, 44 for the -gliadins, 19 for the -gliadins and 15 for the -gliadins. A subset of 333 accessions belonging to fifty populations from Asturias, North of Spain, showed high levels of genetic variation (A = 3.89, P = 0.88, Ne = 3.35 and He = 0.553), indicating that 82.5% of the genetic variation was within populations, and only 18.5% among populations. Thirty-five of these populations presented more of five accessions, in this new subset the values of genetic variation were higher that those of fifty populations (A = 4.49, P = 0.91, Ne = 3.80 and He = 0.595). The genetic variation within populations was 59.7% of the total, and 40.3% among populations, which could be associated to fixation effects of some alleles by genetic drift.     

Intra- and interpopulation diversity for HMW glutenin subunits in Spanish spelt wheat

The diversity of HMW glutenin subunits in spelt wheat, Triticum aestivum ssp. spelta, was studied electrophoretically in 333 accessions grouped in 50 populations originally collected from Asturias, North of Spain, in 1939. The inter- and intra-population distribution of HMW glutenin alleles at the Glu-A1, Glu-B1 and Glu-D1 loci were investigated. The results show that the genetic variation in HMW glutenin subunits is mainly present within populations, being the variation between populations only 21%. The materials analysed showed a wide polymorphism for the HMW glutenin subunits, although some allelic variants were clearly dominant. This suggests the possibility of a loss of variability before the collection that could have increased with the subsequent reduction of the cultivation area of this species in this Spanish region.     

Genetic variation for waxy proteins and amylose content in Spanish spelt wheat (<Emphasis Type="Italic">Triticum spelta</Emphasis> L.)

Spelt wheat is a neglected crop that could be used in the quality breeding of modern common wheat. One important aspect of this quality is the starch composition which is related to the waxy proteins. A collection of 420 accessions of Spanish spelt wheat was analysed for waxy protein composition by SDS–PAGE. Polymorphism was found in the three waxy proteins, detecting differences both in size and in activity, and a new waxy allele (Wx-D1g) was identified. Seed amylose content was also determined and significant differences were detected among the different allelic combinations. In general, the accessions carrying one or two waxy null alleles showed less amylose content. The variation found could be used to enlarge the genetic pool of common wheat, or to develop lines of spelt with different levels of amylose content.     

Characterization of Mexican Creole wheat landraces in relation to morphological characteristics and HMW glutenin subunit composition

One hundred lines derived from 14 wheat landraces collected in Mexico were characterized in relation to spike and grain morphology and HMW-glutenin subunit composition. Up to seven botanical varieties were identified among these materials based in four morphological traits. The remaining nine morphological traits showed wide variation. The allelic variation at the Glu-1 loci was wide, although showed a clear risk of genetic erosion due to the low frequency of some alleles. These genotypes could be used as genetic resources to improve important biotic and abiotic traits as well as to widen genetic diversity controlling the HMW glutenin subunit composition of common wheat.     

Plasma and tissue depletion of florfenicol and florfenicol-amine in chickens

Chickens were used to investigate plasma disposition of florfenicol after single intravenous (i.v.) and oral dose (20 mg kg-1 body weight) and to study residue depletion of florfenicol and its major metabolite florfenicol-amine after multiple oral doses (40 mg kg-1 body weight, daily for 3 days). Plasma and tissue samples were analyzed using a high-performance liquid chromatography (HPLC) method. After i.v. and oral administration, plasma concentration-time curves were best described by a two-compartment open model. The mean [ +/- standard deviation (SD)] elimination half-life (t1/2beta) of florfenicol in plasma was 7.90 +/- 0.48 and 8.34 +/- 0.64 h after i.v. and oral administration, respectively. The maximum plasma concentration was 10.23 +/- 1.67 microg mL-1, and the interval from oral administration until maximal concentration was 0.63 +/- 0.07 h. Oral bioavailability was found to be 87 +/- 16%. Florfenicol was converted to florfenicol-amine. After multiple oral dose (40 mg kg-1 body weight, daily for 3 days), in kidney and liver, concentrations of florfenicol (119.34 +/- 31.81 and 817.34 +/- 91.65 microg kg-1, respectively) and florfenicol-amine (60.67 +/- 13.05 and 48.50 +/- 13.07 microg kg-1, respectively) persisted for 7 days. The prolonged presence of residues of florfenicol and florfenicol-amine in edible tissues can play an important role in human food safety, because the compounds could give rise to a possible health risk. A withdrawal time of 6 days was necessary to ensure that the residues of florfenicol were less than the maximal residue limits or tolerance established by the European Union.     

In Vitro Fertilization (IVF) in Straws and a Short Gamete Coincubation Time Improves the Efficiency of Porcine IVF

The present study was designed to evaluate three different in vitro fertilization (IVF) systems: a straw‐IVF system with 10 min of coincubation, a straw‐IVF system with 6‐h coincubation and the microdrop‐IVF system with 6‐h coincubation (the traditional IVF system used routinely in most of IVF laboratories) in an attempt to reduce polyspermic penetration ( Experiment 1 ). When the straw‐IVF system was tested in combination with two coincubation times, the use of 10 min of coincubation significantly increased (p < 0.001) the penetration rate and the efficiency of fertilization (67.7 ± 6.4% vs 31.9 ± 6.5% and 41.5 ± 2.5% vs 17.6 ± 2.5% for 10 min and 6 h, respectively), while there were no significant differences in the incidence of monospermy between both systems (64.3 ± 5.1% and 67.7 ± 3.4%, for 10 min and 6 h, respectively). The penetration rate in the 6‐h microdrop‐IVF system was higher (93.8 ± 3.6%; p < 0.001) compared with the 10‐min straw‐IVF system (67.7 ± 6.4%), however, monospermy was severely reduced (25.0 ± 4.3% vs 67.7 ± 3.4%, for the 6‐h microdrop‐IVF system and 10‐min straw‐IVF system, respectively). The efficiency of the IVF showed similar values between microdrop and 6‐h straw‐IVF systems, but efficiency was significantly improved (p < 0.05) when the 10‐min straw‐IVF system was used. Experiment 2 was designed to compare porcine in vitro embryo production in two IVF systems, the 6‐h microdrop‐IVF system (1000 sperm per oocyte) and 10‐min straw‐IVF system (30 000 sperm per oocyte). The blastocyst formation rates tended (p = 0.06) to be higher when the 10‐min straw‐IVF system was used compared with the 6‐h microdrop‐IVF system. In addition, the number of total cells per blastocyst increased significantly (p < 0.05) in the 10‐min straw‐IVF system. These results showed that the 10‐min straw‐IVF system is an effective way to decrease polyspermic penetration, and improve the efficiency of fertilization and the quality of blastocysts in terms of cell number per embryo.     

Effects of feed intake on in situ rumen microbial contamination and degradation of feeds

An in situ study was conducted using four rumen cannulated wethers and five feeds (corn grain (CG), soybean meal (SBM), sunflower meal (SFM), vetch–oat hay (VH), and lentil straw (LS)) to determine (using ¹⁵N infusion techniques) the effects of two intake levels (40 and 80 g DM/kg W^0.75) on the microbial contamination and rumen degradation of DM or CP. Degradation studies were also performed for neutral and acid detergent fibres (NDF and ADF, respectively) of VH. In most cases, the evolution with time of the microbial DM adhered to particles fitted well to exponential curves, which diverged between both levels of intake due to the lower values attained at high intake. However, as a consequence of the large and progressive increase of the variability of microbial contamination, statistical evidence is only partial. This behaviour was less evident for CP contamination, especially in forages. The lack of these corrections underestimated (P < 0.001) the effective degradability (ED) of all tested fractions, with important differences between feeds. The intake level had not relevant effects on the distribution of soluble, insoluble available and unavailable fractions of DM or CP (or fibres in VH), but determined some changes in their degradation rates (k_d). Direct relations between these last values and rumen outflow rates (k_p) were shown in some feeds for the degradation of CP and also of DM in concentrates, whereas negative relations were shown for the degradation rates of DM in forages (or NDF and ADF of VH). All ED estimates decreased with the intake rise, but for CP this effect (P < 0.05) was only detected in SBM and VH. These decreases represent 40.8 (CG), 80.8 (SBM), 62.1 (SFM), 66.8 (VH), and 19.5% (LS) than those estimated considering only k_p changes, which shows the interest to consider conjointly the variations of both rates to study the effect of diet changes on ED.