Rapid mobilization of ferritin iron by ascorbate in the presence of oxygen

L-(—)-ascorbate mobilizes iron from horse-spleen ferritin in the presence of oxygen at pH 8.0. The reaction is strongly stimulated by Cu²⁺. Dehydroascorbate and other stable oxidation products of ascorbate are ineffective. We present evidence that monodehydroascorbate mobilizes ferritin iron by reduction.     

Vitrification of germinal-vesicle stage equine oocytes: Effect of cryoprotectant exposure time on in-vitro embryo production

Previous studies have found low rates of blastocyst development (0–11%) after vitrification of germinal vesicle (GV)-stage equine oocytes. In this study, we systematically evaluated a short (non-equilibrating) system for GV-stage oocyte vitrification. In Exp. 1, we assessed oocyte volume in cumulus-oocyte complexes (COCs) exposed to components of a short protocol, using 2% each of ethylene glycol and propylene glycol in the first solution (VS1); 17.5% of each plus 0.3 M trehalose in the second solution (VS2); and fetal bovine serum as the base medium. Based on the time to oocyte minimum volume, we selected a 40-sec exposure to VS1. In Exp. 2, we evaluated exposure times to VS2 and, based on rates of subsequent maturation in vitro, we selected 65 s. In Exp. 3, we used the optimized vitrification system (40-VS1; 65-VS2) and evaluated three warming procedures. Blastocyst development after ICSI was equivalent (15%) for COCs warmed in either standard (trehalose stepwise dilution) or isotonic (base medium) solutions, but was reduced (0%) for COCs warmed in a highly hypertonic (1.5 M trehalose) solution. Exposure to the vitrification and warming solutions, without actual vitrification, was associated with reduced blastocyst development (0–5%; Exp. 4). We conclude that this optimized short protocol supports moderate blastocyst production after vitrification of GV-stage equine COCs. Oocytes can be warmed in isotonic medium, which simplifies the procedure. The systems used still showed a high level of toxicity and further work is needed on both vitrification and warming methods to increase the efficiency of this technique.     

Porcine growth hormone gene expression from viral promoters in transgenic swine

Abstract

The production of porcine growth hormone (pGH) from novel expression vectors containing the promoter/enhancer elements of the Moloney murine leukemia virus (MLV) LTR or the human cytomegalovirus (CMV) immediate early gene was examined in transgenic swine. Both fusion genes resulted in elevated levels of serum pGH, elevation of insulin‐like growth factor 1 (IGF‐1), and a pronounced decrease in carcass fat deposition. The two viral promoter/enhancer elements were constitutively active in the transgenic swine throughout the life of the animals. In individual swine, the CMV‐pGH transgene was expressed predominantly in the pancreas while the MLV‐pGH transgene was expressed in a wide variety of tissues. These swine were infertile, had insulin resistance, and demonstrated an accelerated form of osteochondritis dissicans. Our results show that excess pGH produces a phenotype identical to that seen in swine expressing heterologous growth hormones, and provides a baseline for assessing the overall efficiency of producing transgenic swine. Furthermore, our data suggests that unregulated pGH production, even at 15 times normal levels and independent of the tissue source, has adverse effects that outweigh the desired reduction in carcass fat deposition in transgenic swine.     

Distribution of purinergic P2X receptors in the equine digit, cervical spinal cord and dorsal root ganglia

Purinergic pathways are considered important in pain transmission, and P2X receptors are a key part of this system which has received little attention in the horse. The aim of this study was to identify and characterise the distribution of P2X receptor subtypes in the equine digit and associated vasculature and nervous tissue, including peripheral nerves, dorsal root ganglia and cervical spinal cord, using PCR, Western blot analysis and immunohistochemistry. mRNA signal for most of the tested P2X receptor subunits (P2X_{1–5, 7}) was detected in all sampled equine tissues, whereas P2X₆ receptor subunit was predominantly expressed in the dorsal root ganglia and spinal cord. Western blot analysis validated the specificity of P2X_{1–3, 7} antibodies, and these were used in immunohistochemistry studies. P2X_{1–3, 7} receptor subunits were found in smooth muscle cells in the palmar digital artery and vein with the exception of the P2X₃ subunit that was present only in the vein. However, endothelial cells in the palmar digital artery and vein were positive only for P2X₂ and P2X₃ receptor subunits. Neurons and nerve fibres in the peripheral and central nervous system were positive for P2X_1–3 receptor subunits, whereas glial cells were positive for P2X₇ and P2X_{1 and 2} receptor subunits. This previously unreported distribution of P2X subtypes may suggest important tissue specific roles in physiological and pathological processes.     

Faecal egg counts and nemabiome metabarcoding highlight the genomic complexity of equine cyathostomin communities and provide insight into their dynamics in a Scottish native pony herd

Understanding the composition of gastrointestinal nematode communities may help to mitigate or exploit parasite adaptations within their host. We have used nemabiome deep amplicon sequencing of internal transcribed spacer-2 (ITS-2) ribosomal DNA to describe the temporal and host species composition of gastrointestinal nematode communities following sampling of six Scottish ponies across 57 months. In the absence of parasite control, each horse showed seasonal trends of increases and decreases in faecal egg counts, consistent with the epidemiology of equine strongylid parasites, however, the composition of parasites within individuals changed over time. Sixteen presumptive strongylid species were identified in each of the horses, 13 of which were distributed in a complex clade together with small numbers of amplicon sequences which could not be classified beyond the Cyathostominae subfamily level. Egg shedding of seven trichostrongylid species, which had previously been identified in co-grazed Soay sheep, was identified during the early spring. Faecal egg counts and the percentage of amplicon sequences assigned to each gastrointestinal nematode species were combined to describe their relative abundance across both host and time. Significant differences in species diversity between horses and between months were observed, being greatest from March to May and least from October to December. The magnitude of the individual horse effect varied between months and, conversely, the magnitude of the seasonal effect varied between individual horses. The most abundant gastrointestinal nematode in each of the horses was Cylicostephanus longibursatus (46.6% overall), while the abundance of the other strongylid species varied between horses and relative to each other. Patent C. longibursatus infections over the winter months might represent a genetic adaptation towards longer adult worm survival, or a lower rate of developmental arrest in the autumn. This study provides insight into highly complex phylogenetic relationships between closely related cyathostomin species; and describes the dynamics of egg shedding and pasture contamination of co-infecting equine gastrointestinal nematode communities. The results could be applied to determine how climatic and management factors affect the equilibrium between hosts and their parasites, and to inform the development of sustainable gastrointestinal nematode control strategies for different host species.     

PMab-219: A monoclonal antibody for the immunohistochemical analysis of horse podoplanin

Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine podoplanin (PDPN), a lymphatic endothelial cell marker, have been established in our previous studies. However, mAbs against horse PDPN (horPDPN), which are useful for immunohistochemical analysis, remain to be developed. In the present study, mice were immunized with horPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/horPDPN), and hybridomas producing mAbs against horPDPN were screened using flow cytometry. One of the mAbs, PMab-219 (IgG_2a, kappa), specifically detected CHO/horPDPN cells via flow cytometry and recognized horPDPN protein using Western blotting. Furthermore, PMab-219 strongly stained CHO/horPDPN via immunohistochemistry. These findings suggest that PMab-219 is useful for investigating the function of horPDPN.     

Selection signatures in Shetland ponies

Shetland ponies were selected for numerous traits including small stature, strength, hardiness and longevity. Despite the different selection criteria, Shetland ponies are well known for their small stature. We performed a selection signature analysis including genome‐wide SNPs of 75 Shetland ponies and 76 large‐sized horses. Based upon this dataset, we identified a selection signature on equine chromosome (ECA) 1 between 103.8 Mb and 108.5 Mb. A total of 33 annotated genes are located within this interval including the IGF1R gene at 104.2 Mb and the ADAMTS17 gene at 105.4 Mb. These two genes are well known to have a major impact on body height in numerous species including humans. Homozygosity mapping in the Shetland ponies identified a region with increased homozygosity between 107.4 Mb and 108.5 Mb. None of the annotated genes in this region have so far been associated with height. Thus, we cannot exclude the possibility that the identified selection signature on ECA1 is associated with some trait other than height, for which Shetland ponies were selected.     

Large herbivores as mobile links between isolated nature reserves through adhesive seed dispersal

Question: Do large herbivores contribute to the dispersal of plant seeds between isolated habitats by epizoochory? Location: Nature reserves in Flanders, Belgium. Methods: Epizoochory was studied by brushing plant seeds from the fur of 201 domesticated large herbivores (Galloway cattle, donkeys and horses), grazing in 27 Flemish nature reserves. Several herbivores were examined after transport between different nature reserves as part of the seasonal grazing system in Flanders, allowing detection of seed dispersal both within and between reserves. The seedling emergence method was used to identify the dispersed plant species. Results: In total, 6385 epizoochorous seeds from 75 species germinated, yet the real seed quantity was underestimated by the seedling emergence method. A wide variety of seed morphology, seed weights and plant heights was represented among the dispersed species, many of which had a transient seed bank. There was a gradual turnover in epizoochorous species composition in the course of the vegetation season, and seed dispersal occurred both within and between different nature reserves. Conclusions: Domesticated large herbivores, as models for wild mammals in the present and the past, are important dispersers of many plant species. Through seasonal grazing, the herbivores function as ‘mobile link organisms’, connecting isolated nature reserves through seed dispersal, possibly influencing vegetation development and long‐term survival of plant populations. As such, large herbivores are important instruments in ecological restoration, especially in fragmented ecosystems.     

A preliminary study of the effects of handling type on horses’ emotional reactivity and the human–horse relationship

Handling is a crucial component of the human–horse relationship. Here, we report data from an experiment conducted to assess and compare the effect of two training methods. Two groups of six Welsh mares were trained during four sessions of 50 min, one handled with traditional exercises (halter leading, grooming/brushing, lifting feet, lunging and pseudo-saddling (using only girth and saddle pad) and the second group with natural horsemanship exercises (desensitization, yielding to body pressure, lunging and free-lunging). Emotional reactivity (ER) and the human–horse relationship (HHR) were assessed both prior to and following handling. A social isolation test, a neophobia test and a bridge test were used to assess ER. HHR was assessed through test of spontaneous approach to, and forced approach by, an unknown human.Horses’ ER decreased after both types of handling as indicated by decreases in the occurrence of whinnying during stressful situations. Head movement (jerk/shake) was the most sensitive variable to handling type. In the spontaneous approach tests, horses in the traditional handling group showed higher latencies to approach a motionless person after handling than did the natural horsemanship group. Our study suggests that natural horsemanship exercises could be more efficient than traditional exercises for improving horses’ HHR.