Potentiation of triclabendazole action in vivo against a triclabendazole-resistant isolate of Fasciola hepatica following its co-administration with the metabolic inhibitor, ketoconazole

An in vivo study in the laboratory rat model has been carried out to monitor morphological changes in adult Fasciola hepatica over a 4-day period resulting from co-treatment with triclabendazole (TCBZ) and ketoconazole (KTZ), a cytochrome P450 inhibitor. Rats were infected with the triclabendazole-resistant Oberon isolate of F. hepatica, dosed orally with triclabendazole at a dosage of 10 mg/kg live weight and ketoconazole at a dosage of 10 mg/kg live weight. Flukes were recovered at 24, 48, 72 and 96 h post-treatment (p.t.) and changes to fluke ultrastructure were assessed using transmission electron microscopy (TEM). Results showed an increase in the severity of changes to the fluke ultrastructure with time p.t. Swelling of the basal infolds and the associated mucopolysaccharide masses became more severe with time. Golgi complexes, if present, were greatly reduced in size and number by 96 h p.t., and sub-tegumental flooding was seen from the 72 h time-period onwards. Some sloughing of the tegumental covering over the spines was observed at 96 h p.t. The results demonstrated that the Oberon isolate is more sensitive to TCBZ action in the presence of KTZ than to TCBZ alone, reinforcing the idea that altered drug metabolism is involved in the resistance mechanism. Moreover, they support the concept that TCBZ + inhibitor combinations (aimed at altering drug pharmacokinetics and potentiating the action of TCBZ) could be used in the treatment of TCBZ-R populations of F. hepatica.     

Influence of the Cumulus and Gonadotropins on the Metabolic Profile of Porcine Cumulus–Oocyte Complexes During In Vitro Maturation

The aim of this work was to examine the influence of the cumulus and gonadotropins on the metabolic profile of porcine cumulus oocyte complexes (COCs) during in vitro maturation. Immature COCs were assigned to morphological classes A₁ (with a dense cumulus), A₂ (with a translucent cumulus), B₁ (with the corona radiata), B₂ (with only some remaining cumulus cells) and matured with or without gonadotropins. Glycolysis and ammonia production were higher in the A class COCs; gonadotropins increased both, especially in the A₁ COCs (p < 0.05). The A class COCs had the highest initial protein contents and at the end of in vitro maturation. Furthermore, hormonal stimulation induced a similar increase in protein contents of both A classes (p < 0.05). The neutral lipid content and reactive oxygen species (ROS) levels were similar in the immature oocytes of the COCs of all classes. A reduction was seen in both these variables when maturation proceeded either in the presence or absence of gonadotropins. The cumulus type surrounding the oocyte is related to the metabolism of carbohydrates and amino acids by the COC during in vitro maturation under gonadotropic stimulation. Oocyte lipolytic activity and ROS production appear to be independent of the surrounding cumulus and the presence of gonadotropins.     

Relationship between increased albendazole systemic exposure and changes in single nucleotide polymorphisms on the β-tubulin isotype 1 encoding gene in Haemonchus contortus

Resistance to benzimidazole anthelmintics in the nematode Haemonchus contortus has been correlated with single nucleotide polymorphisms (SNPs) on the β-tubulin isotype 1 gene. Three mutations can be used as markers for the detection of resistance, namely SNPs at position 200 and 167 (both TTC to TAC) or at position 198 (GAA to GCA). Harbouring a resistance genotype at any one of these codons can lead to a resistant phenotype. Our objective in this study was to analyse the frequencies of the three mutations when the albendazole dose rate and selection pressure were increased. We used adult H. contortus (males and females) collected directly from the abomasum of untreated lambs, or lambs treated with the manufacturer's recommended dose rate (5mg/kg), three times the recommended dose rate (15 mg/kg), or nine times the recommended dose rate (45 mg/kg). Anthelmintic efficacy was determined by worm and egg count reductions. For the surviving worms of the four treatment groups, the frequencies of each resistance SNP at codons 167, 200 and 198 were measured using pyrosequencing. Our results showed a strong relationship between an increasing dose rate and an increase in the frequency of the (TAC)(200) SNP and a decrease in the (TAC)(167) SNP. All worms genotyped were GAA at codon 198.     

Histochemical and Morphometric Analyses of the Musculature of the Forelimb of the Subterranean Rodent Ctenomys talarum (Octodontoidea)

Histochemical and morphometric analyses were performed to characterize the fibre‐type composition of two forelimb muscles of the South American subterranean rodent Ctenomys talarum. The studied muscles were the triceps lateralis, an extensor of the elbow, and the teres major, a flexor of the shoulder. It was found that these muscles had an elevated proportion of fast oxidative‐glycolytic (FOG) fibres, and lower proportions of slow oxidative (SO) and fast glycolytic (FG) fibres. This composition probably qualifies the teres major and triceps muscles to perform the sustained effort required in tunnelling excavation. The results were discussed considering published data on fibre‐type composition of mammals having different modes of life and digging behaviour. We here suggest that C. talarum has the potential of generating forces linked to rapid, powerful movements during sustained periods by means of an elevated proportion of FOG fibres, together with osteological changes that result in a great mechanical advantage of the forelimb muscles.     

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.     

Oestrogen,Progesterone and Oxytocin Receptors and COX‐2 Expression in Endometrial Biopsy Samples from the Induction of Ovulation to Luteolysis in Llamas (Lama glama)

Endometrial expression of oestrogen (ERα), progesterone (PR) and oxytocin receptor (OR) and cyclooxygenase‐2 (COX‐2) was evaluated from the induction of ovulation to luteolysis in llamas. Ovarian activity was daily assessed by ultrasonography in five females. Ovulation was induced immediately after the detection of an ovulatory follicle by a GnRH injection (Day 0). Endometrial samples were obtained by transcervical biopsies from the left and right horns on day 0 and days 4, 8, 10 and 12 post‐GnRH. Blood samples were collected daily for progesterone and estradiol‐17β determinations by RIA. An immunohistochemical technique was used to study receptors population and COX‐2 expression which were then evaluated by two independent observers. The expression of ERα and PR was highest on day 0 in the luminal epithelium and stroma in association with high plasma estradiol‐17β concentrations. Thereafter, a decrease in ERα population was registered on day 4 and a new increase of its expression was observed between days 8 and 12 in those cell types. Conversely, PR population was gradually down‐regulated until its lowest expression was reached on day 10 post‐GnRH in the luminal epithelium. Content of OR was similar throughout the study in all cell types. The expression of COX‐2 was highest from day 8 to 12 post‐GnRH in the luminal epithelium, in relation to the time of maximal PGF_2α release. Both steroid receptors populations and COX‐2 expression were similar between horns. Meanwhile, OR expression was higher in the right than in the left uterine horn. In summary, this study showed that the loss of endometrium sensitivity to progesterone by days 8–10 post‐induction of ovulation and the concomitant increase of COX‐2 expression could play a key role in the mechanism of luteolysis and somehow be related to the short corpus luteum lifespan of llamas.     

Development of a stand density management diagram for even-aged pedunculate oak stands and its use in designing thinning schedules

Density management is the usual method used by silviculturiststo achieve a desired future stand condition, and one of themost effective methods of design, display and evaluation ofalternative density management regimes in even-aged stands isthe use of stand density management diagrams. In the presentstudy, we describe a method for developing thinning schedulesfor even-aged pedunculate oak (Quercus robur L.) stands in Galicia(north-western Spain), using a density management diagram. Thediagram integrates the relationships among stand density, dominantheight, quadratic mean diameter and stand volume in a singlegraph. The data used in its construction were obtained from172 sample plots located throughout Galicia. The diagram isbasically composed of two equations: the first relates the quadraticmean diameter to the stand density and dominant height; thesecond relates the stand volume to the quadratic mean diameter,stand density and dominant height. These equations were fittedsimultaneously using full information maximum likelihood. Therelative spacing index is used to characterize the growing stockand the diagram provides isolines for dominant height, numberof trees per hectare, quadratic mean diameter and stand volume.Dominant height isolines together with the site index curvesallow specification of the timing of thinnings while intermediateand final harvest volumes are calculated using the stand volumesisolines.     

Predictive relationship between polyphenol and nonfat cocoa solids content of chocolate

Chocolate is often labeled with percent cocoa solids content. It is assumed that higher cocoa solids contents are indicative of higher polyphenol concentrations, which have potential health benefits. However, cocoa solids include polyphenol-free cocoa butter and polyphenol-rich nonfat cocoa solids (NFCS). In this study the strength of the relationship between NFCS content (estimated by theobromine as a proxy) and polyphenol content was tested in chocolate samples with labeled cocoa solids contents in the range of 20-100%, grouped as dark (n = 46), milk (n = 8), and those chocolates containing inclusions such as wafers or nuts (n = 15). The relationship was calculated with regard to both total polyphenol content and individual polyphenols. In dark chocolates, NFCS is linearly related to total polyphenols (r2 = 0.73). Total polyphenol content appears to be systematically slightly higher for milk chocolates than estimated by the dark chocolate model, whereas for chocolates containing other ingredients, the estimates fall close to or slightly below the model results. This shows that extra components such as milk, wafers, or nuts might influence the measurements of both theobromine and polyphenol contents. For each of the six main polyphenols (as well as their sum), the relationship with the estimated NFCS was much lower than for total polyphenols (r2 < 0.40), but these relationships were independent of the nature of the chocolate type, indicating that they might still have some predictive capabilities.