Melatonin reduces apoptotic cells,SOD2 and HSPB1 and improves the in vitro production and quality of bovine blastocysts

Effects of adding different concentrations of melatonin (10^?7, 10^?9 and 10^?11 M) to maturation (Experiment 1; Control, IVM  + 10^?7, IVM  + 10^?9, IVM  + 10^?11) and culture media (Experiment 2; Control, IVC  + 10^?7, IVC  + 10^?9, IVC  + 10^?11) were evaluated on in vitro bovine embryonic development. The optimal concentration of melatonin (10^?9 M) from Experiments 1–2 was tested in both maturation and/or culture media of Experiment 3 (Control, IVM  + 10^?9, IVC  + 10^?9, IVM /IVC  + 10^?9). In Experiment 1, maturated oocytes from Control and IVM  + 10^?9 treatments showed increased glutathione content, mitochondrial membrane potential and percentage of Grade I blastocysts (40.6% and 43%, respectively). In Experiment 2, an increase in the percentage of Grade I blastocysts was detected in IVC  + 10^?7 (43.5%; 56.7%) and IVC  + 10^?9 (47.4%; 57.4%). Moreover, a lower number and percentage of apoptotic cells in blastocysts were observed in the IVC  + 10^?9 group compared to Control (3.8 ± 0.6; 3.6% versus 6.1 ± 0.6; 5.3%). In Experiment 3, the IVC  + 10^?9 treatment increased percentage of Grade I blastocysts with a lower number of apoptotic cells compared to IVM /IVC  + 10^?9 group (52.6%; 3.0 ± 0.5 versus 46.0%; 5.4 ± 1.0). The IVC  + 10^?9 treatment also had a higher mRNA expression of antioxidant gene (SOD 2) compared to the Control, as well as the heat shock protein (HSPB 1) compared to the IVM  + 10^?9. Reactive oxygen species production was greater in the IVM /IVC  + 10^?9 treatment group. In conclusion, the 10^?9 M concentration of melatonin and the in vitro production phase in which it is used directly affected embryonic development and quality.     

Liver biopsy techniques for adult horses and neonatal foals to assess copper status

Objective To evaluate standing, percutaneous, ultrasound-guided, transthoracic liver biopsy in mares, and transabdominal, laparoscopically-guided, liver biopsy under general anaesthesia in foals, as techniques for obtaining tissue for assessment of copper status. The techniques were evaluated with respect to ease of use and effect on the animal.
Procedure Twenty of 24 Thoroughbred mares and 21 of their foals were biopsied. The animals were part of a larger study of the effect of copper supplementation on copper status and the prevalence of developmental orthopaedic disease. Livers were also collected from unrelated horses and sampled to investigate the variability in the distribution of copper in liver tissue.
Result The biopsy technique caused no lasting effect on the mares, but there was an increased risk of viscus penetration associated with taking multiple biopsy cores. The use of ultrasonography to scan the target area for the liver identified four cases that were not appropriate candidates for liver biopsy, because of large intestine being located in the biopsy area. In the foals there were no serious postoperative adverse effects, nor was there any evidence of problems caused by the procedure when the abdomen was examined post-mortem at 5 months of age. In livers collected to investigate the variability of copper concentration, copper appeared to be relatively evenly distributed through the liver.
Conclusion Standing, percutaneous, ultrasound-guided, transthoracic liver biopsy in mares, and transabdominal, laparoscopically-guided, liver biopsy under general anaesthesia in foals are convenient procedures for obtaining liver tissue for assessing copper status in horses. The use of ultrasound to identify liver tissue is recommended, especially in older mares.     

Localized Melanization of Appressoria Is Required for Pathogenicity of Venturia inaequalis

ABSTRACT During formation of appressoria produced from conidia and ascospores of Venturia inaequalis, a dark brown ring structure was detected at the base of appressoria. This melanized appressorial ring structure (MARS) was attached to the leaf surface like a sealing ring and formed the fungus-plant interface; it is believed to be required for pathogen penetration of the cuticle. Neither germ tubes nor infection structures beneath the cuticle were found to be visibly melanized. MARS were formed not only on apple leaves but also on leaves of nonhost plants and artificial surfaces differing in hydrophobicity; the formation of appressoria and MARS was confined to hard surfaces. The melanin nature of the ring was confirmed by using melanin biosynthesis inhibitors. Applications prior to inoculation largely inhibited the melanization and reduced infection rate by 45 to 80%; curative applications were not effective. Transmission electron microscopy verified a localized melanization of the cell wall around the penetration pore, and melanin was incorporated into all layers of the fungal cell wall. Appressoria without MARS were not able to infect the plant, suggesting that this structure can be considered to be a pathogenicity factor in V. inaequalis.     

Rhodococcus equi-associated osteomyelitis in foals

Two cases of Rhodococcus equl infection in foals are described, in which osteomyelitis was a feature. Because rhodococcal infection is usually low grade and chronic, and because the signs of early metaphysitis can be subtle, any articular or periarticular swelling in a foal from a farm with a history of rhodococcosis should be strongly suspected to be associated with R equl until proven otherwise.     

A survey of feeding,management and faecal pH of Thoroughbred racehorses in the North Island of New Zealand

AIM: To identify feeding and management variables associated with variation in faecal pH within a population of intensively managed Thoroughbred racehorses in New Zealand.

METHODS: A cross-sectional survey was conducted of 16 racehorse trainers in the North Island of New Zealand. Interviews were conducted at the trainers' stables to obtain information on feeding and management of horses, and faecal samples were collected and faecal pH measured.

RESULTS: Ninety-seven percent of the horses surveyed were confined in an area ≤5 × 5 m for ≥12 h/day. Trainer's age, number of years they had trained horses, age and gender of horses, weeks in race training, racing class, frequency of feeding, bedding type, and exercise workload had no effect on mean faecal pH. Acidic faecal pH (pH ≤6.32) was associated with stables with ≤12 horses, and trainers at stables with ≤12 horses offered more concentrate feed than those at stables with >12 horses. Acidic faecal pH was associated with trainers who offered 4 kg of grain as the only form of concentrate fed, or offered ≤2.25 kg hay/day. Horses that displayed stable vices had less acidic faecal pH than horses that did not display stable vices, viz pH 6.70 (standard error of the mean (SEM) 0.135) vs 6.43 (SEM 0.029) (p=0.04).

CONCLUSIONS AND CLINICAL RELEVANCE: Racehorse management in New Zealand is similar to that observed in other major racing countries. Trainers with ≤12 horses fed more concentrates and their horses had lower faecal pH than those of trainers with >12 horses. Irrespective of management system, it appears important to provide at least 2.25 kg of hay/day ad libitum, to buffer hindgut acidosis associated with diets high in soluble carbohydrate.     

Musculoskeletal responses of 2-year-old Thoroughbred horses to early training. 8. Quantitative back-scattered electron scanning electron microscopy and confocal fluorescence microscopy of the epiphysis of the third metacarpal bone

AIM: To characterise and explain the increase in density evident by computerised tomography (CT) and radiography in companion studies as a response to training, in bone in the palmar and dorsal regions of the condyles of the third metacarpal bone (Mc3) of 2-year-old Thoroughbred horses.

METHODS: Compositional back-scattered electron (BSE) imaging in scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM) were conducted on polymethyl methacrylate (PMMA)-embedded mediolateral slices of the right distal Mc3 from seven 2-year-old Thoroughbred horses trained on a racetrack and seven untrained horses kept at pasture. One left Mc3 from each group was studied in transverse section planes. This study focussed on regions of Mc3 found to differ in density between the trained and untrained horses in companion studies using CT and radiography.

RESULTS: The increase of bone density in the condyles of Mc3 in trained horses compared with untrained horses occurred, without prior osteoclastic resorption, via the deposition of new bone on pre-existing internal surfaces. Within prior marrow spaces of cancellous bone, there was also rapid formation of immature strands and fronds of bone which were more cellular and mineralised, and more lamellar bone tissue was deposited on these new scaffolding elements in the trained horses. Both resulted in increased bone volume fraction (BVF). The microscopic mineralisation density of the bulk of the new tissue was lower than in pre-existing bone, and CT and radiography underestimated the increase in BVF. The new tissue was thus probably less stiff at the microscopic scale than pre-existing bone, though its addition would stiffen the global structure.

CONCLUSIONS: In Mc3 of all the trained horses, there were obvious differences in microscopic structure compared with those from the untrained horses. Moderate, industry-standard levels of exercise used to prepare young horses for racing induced the formation of new bone in non-bone spaces in bone tissue, such that the bone organ should better withstand later increased levels of exercise.     

Musculoskeletal responses of 2-year-old Thoroughbred horses to early training. 1. Study design,and clinical,nutritional, radiological and histological observations

AIMS: This is the first in a series of papers reporting studies in 2-year-old Thoroughbred racehorses that aimed to determine the response of musculoskeletal tissues to early training on grass and sand racetracks. In this paper, the experimental set-up of the whole study is described, and nutritional, workload, and clinical, radiographic and pathological outcomes are reported, including semi-quantitative assessment of macroscopic changes in articular cartilage.

METHODS: The study group comprised 14 two-year-old Thoroughbred fillies reared entirely at pasture. Of these, seven were selected by a licensed racehorse trainer to undergo a 4-week period of initial training in which they were taught to accept saddle and rider, followed by a 13-week period of flatrace training at a racetrack (Weeks 1–13); the other seven fillies were confined to large grass enclosures and were not trained. Nutrient, including macro- and trace-element intakes were estimated. Distances cantered or galloped and average velocities were quantified for the trained horses. All horses were observed daily, weighed approximately weekly, and underwent a clinical lameness examination at Weeks 5, 9 and 13. Distal forelimbs were radiographed prior to Week 1, during Weeks 7–8, and again at the end of the study, when macroscopic changes in articular cartilage of the proximal surface of the proximal phalanx were also scored after staining with India ink.

RESULTS: Dietary intakes met or exceeded recommended requirements for all nutrients except sodium, which was low in the trained horses. Bodyweight increased throughout the study in the untrained horses, and increased until Week 7 and then decreased slightly in the trained horses. Mean velocity data were used to define three stages of the training programme: Stage 1 comprised canter in Weeks 1–4; Stage 2 comprised canter in Weeks 5–8; and Stage 3 comprised canter in Weeks 9–13 and galloping twice weekly. Four of seven horses completed training. These covered a mean distance of 179.2 km at mean velocities (excluding gallops) of 7.63 m/sec (SD 0.58), 8.99 m/sec (SD 0.56), and 8.43 m/sec (SD 0.74) for Stages 1–3, respectively, and galloped 4.45 km at 14.4 (SD 0.1) m/sec. The three horses that did not complete training became lame in Weeks 9, 10 and 11, and covered 147.9 km at velocities of 7.38 m/sec (SD 0.44), 8.88 m/sec (SD 0.33) and 8.43 m/sec (SD 0.59) and galloped 2.1 km. Overall, slight or intermittent lameness in trained horses was noted on 76/655 (12%) of horse observation days. Swelling was evident on 284/655 (41%) of horse observation days in the metacarpophalangeal (MCPJ) and metatarsophalangeal (MTPJ) joints (92%), palmar metacarpal tendon region (7%) or carpus (1%). Swelling of the MCPJ or MTPJ was not associated with obvious lameness. Radiographic changes were minor and no gross lesions in bone or tendon tissue were evident except for one case of dorsal metacarpal disease. Post mortem, the cartilage of some MCPJ and MTPJ had obvious wear lines and high lesion scores, which were not consistently related to clinical evidence of pain, lameness or joint swelling. Mean lesion scores were not significantly different between the MCPJ and MTPJ, or between trained and untrained horses.

CONCLUSIONS: Workload can be readily quantified in racehorses under semi-commercial training conditions. Obvious lesions in cartilage of the MCPJ or MTPJ were present in some trained and some untrained horses and not consistently associated with clinical evidence of lameness, joint swelling or change in other connective tissues.

CLINICAL RELEVANCE: Workload data in racing horses are likely to be highly relevant for studying the pathogenesis of changes in bone, tendon and cartilage during training, for training management and for risk analysis in racehorse populations. Although obvious cartilage lesions produced little clinical effect, such lesions have previously been shown to be progressive and to prejudice athletic capability. Detection of such occult lesions in young horses will require more sophisticated detection methods.     

Automated identification of sugar beet diseases using smartphones

Cercospora leaf spot (CLS) poses a high economic risk to sugar beet production due to its potential to greatly reduce yield and quality. For successful integrated management of CLS, rapid and accurate identification of the disease is essential. Diagnosis on the basis of typical visual symptoms is often compromised by the inability to differentiate CLS symptoms from similar symptoms caused by other foliar pathogens of varying significance, or from abiotic stress. An automated detection and classification of CLS and other leaf diseases, enabling a reliable basis for decisions in disease control, would be an alternative to visual as well as molecular and serological methods. This paper presents an algorithm based on a RGB‐image database captured with smartphone cameras for the identification of sugar beet leaf diseases. This tool combines image acquisition and segmentation on the smartphone and advanced image data processing on a server, based on texture features using colour, intensity and gradient values. The diseases are classified using a support vector machine with radial basis function kernel. The algorithm is suitable for binary‐class and multi‐class classification approaches, i.e. the separation between diseased and non‐diseased, and the differentiation among leaf diseases and non‐infected tissue. The classification accuracy for the differentiation of CLS, ramularia leaf spot, phoma leaf spot, beet rust and bacterial blight was 82%, better than that of sugar beet experts classifying diseases from images. However, the technology has not been tested by practitioners. This tool can be adapted to other crops and their diseases and may contribute to improved decision‐making in integrated disease control.     

Thermographic assessment of scab disease on apple leaves

Phytopathogenic fungi may affect both the cuticular and the stomatal conductance of plant tissue resulting in significant modifications of leaf temperature. Venturia inaequalis colonizes apple leaves below the cuticle (subcuticularly) causing scab disease. The suitability of digital infrared thermography for sensing and quantifying apple scab was assessed by investigating the effects of V. inaequalis on the water balance of apple leaves in relation to the disease stage and the severity of scab. Transpiration was measured by infrared thermo-imaging to evaluate spatial heterogeneity of the leaves in response to localized infections. Fungal development was assessed microscopically. Subcuticular growth of the pathogen caused localized decreases in leaf temperature before symptoms appeared that significantly increased the maximum temperature difference (MTD) of leaves. The MTD increased with scab development and was strongly correlated to the size of infection sites (r2_linear = 0.85) and overall disease severity (% diseased leaf area, r2_square = 0.71). In later stages of the disease, the MTD decreased because of leaf senescence. Thermographic measurements revealed differences in disease severity resulting from disease stage, resistance of host tissue and differences in the aggressiveness of V. inaequalis isolates. Subcuticular growth of the pathogen was beyond the area of conidia production, therefore, the area of leaf with increased transpiration was larger than the scab lesions; the proportion decreased from >70% in the early stages to <20% for mature lesions. Leaf transpiration was increased by all stages of scab development, therefore, MTD may be used not only for the differentiation between diseased and non-diseased leaves, but also for disease quantification, e.g. in screening systems and monitoring in precision agriculture.