Oestradiol Induced Inhibition of Neuroendocrine Marker Expression in Leydig Cells of Adult Rats

The objectives of this work were to determine the changes in the expression of neuroendocrine markers in Leydig cell by oestradiol treatment, and to determine whether testosterone is able to recover partially the effects of hormonal suppression induced by oestradiol. Adult male rats were injected daily with either 50 microg of oestradiol or oestradiol plus testosterone propionate (25 mg every 3 days) for 15 days. The animals were sacrificed and testicles were dissected and processed by routine histological protocols. FSH and LH serum levels were determined by radioimmunoassay. The visualization of antigens was achieved by the streptavidin-peroxidase immunohistochemical method. Antibodies against chromogranin A (CrA), S-100 protein (S-100), P substance (PS), synaptofisin (SYN), neurofilament protein (NF), gliofibrillary acidic protein (GFAP) and neuron specific enolase (NSE) were used. The mean LH and FSH serum concentrations were consistently suppressed with hormonal treatments. Intermediate filaments (NF and GFAP) showed no difference in their expression. The expression of S-100, NSE and SYN was significantly lower in both hormone-treated groups. In oestradiol-treated rats, the immunoreactivity of CrA and SP decreased significantly but was restored after testosterone supplementation. Although the nature and functions of many of these substances in Leydig cells remain unknown, these results are consistent with the hypothesis that the expression of some neuroendocrine markers is hormonally controlled.     

Flooding tolerance of spelt (Triticum spelta L.) compared to wheat (Triticum aestivum L.) – A physiological and genetic approach

In marginal, agroclimatic zones, yield is often affected by flooding, but the effect is much less for winter spelt (Triticum spelta L.) than for winter wheat (Triticum aestivum L.). This study evaluates the reaction of a wheat x spelt population (F₅ RILs of Forno x Oberkulmer) to flooding stress in the early phase of germination. Lines with greater tolerance to 48 h flooding just after imbibition showed less electrolyte leakage (r = -0.79) indicating greater membrane integrity and better survival. Five QTL explaining 40.6% of the phenotypic variance for survival to flooding were found, and localized on the chromosomes 2B, 3B,5A, and 7S. The tolerance to 48 h flooding four days after sowing was best correlated with the mean germination time (r = 0.8), indicating that the plants with a fast coleoptile growth during flooding are less susceptible to flooding. Ten QTL were found for seedling growth index after flooding explaining 35.5% of the phenotypic variance. They were localized on chromosomes 2A, 2B, 2D, 3A, 4B, 5A, 5B, 6A, and 7S. Standard varieties of spelt and wheat showed the same tolerance characteristics. The possibility to use marker assisted selection for flooding tolerance is discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Hypoglycemic effect of lupin seed γ-conglutin in experimental animals and healthy human subjects

A lupin seed γ-conglutin-enriched preparation was tested in a glucose overload trial with both murine models and adult healthy volunteers. The results with rats showed a dose-dependent significant decrease of blood glucose concentration, which confirmed previous findings obtained with the purified protein. Moreover, three test-product doses equivalent to 630, 315, and 157.5 mg γ-conglutin, orally administered 30 min before the carbohydrate supply, showed a relevant hypoglycemic effect in human trials. Insulin concentrations were not significantly affected. The general hematic parameters did not change at all.This is the first report on the glucose-lowering effect of lupin γ-conglutin in human subjects.     

Linoleic acid increases adhesion,chemotaxis, granule release,intracellular calcium mobilisation,MAPK phosphorylation and gene expression in bovine neutrophils

Neutrophils are critical to the innate immune response; therefore, the proper function of neutrophils is critical to avoid the development of certain diseases. Linoleic acid, a polyunsaturated long-chain fatty acid, is one of the most abundant long-chain fatty acids found in the plasma of cows after giving birth. In this study, we evaluated the effects of linoleic acid treatment on bovine neutrophil adhesion, chemotaxis, metalloproteinase (MMP)-9 release, CD11b expression, intracellular calcium mobilisation, mitogen-activating protein kinase (MAPK) phosphorylation and COX-2 and IL-8 expression. Bovine neutrophils isolated from healthy heifers were incubated with different concentrations of linoleic acid, and then neutrophil responses were evaluated. Our results show that the treatment of neutrophils with 100 μM linoleic acid increased their adhesion to the bovine endothelial cell line CPA47. The results of a transwell migration assay revealed that linoleic acid could also promote the chemotaxis of bovine neutrophils. Furthermore, linoleic acid treatment increased MMP-9 activity and CD11b cell surface expression in neutrophils. Fifty and 100 μM linoleic acid also increased intracellular calcium mobilisation in neutrophils loaded with Fluo-4 AM dye. Linoleic acid also rapidly (2–5 min) stimulated the phosphorylation of ERK1/2 and p38 MAPK as evaluated by immunoblot. Finally, COX-2 and IL-8 mRNA expression increased after 2 h of linoleic acid treatment. In conclusion, linoleic acid stimulates adhesion, chemotaxis, granule release and intracellular responses in bovine neutrophils.     

Mucopolysaccharidosis type VI in a Miniature Poodle-type dog caused by a deletion in the arylsulphatase B gene

Abstract

AIM: To investigate and characterise an inborn error of metabolism in a dog with skeletal and ocular abnormalities.

METHODS: A 2.5-year-old small male Miniature Poodle-like dog was presented with gross joint laxity and bilateral corneal opacities. Clinical examination was augmented by routine haematology, serum chemistry, radiographs, pathology, enzymology and molecular genetic studies. Euthanasia was requested when the dog was 3 years of age because of progressively decreasing quality of life.

RESULTS: Radiology revealed generalised epiphyseal dysplasia, malformed vertebral bodies, luxation/subluxation of appendicular and lumbosacral joints with hypoplasia of the odontoid process and hyoid apparatus. These clinical and radiographic findings, together with a positive urinary Berry spot test for mucopolysaccharides, and metachromatic granules in leucocytes, were indicative of a mucopolysaccharidosis (MPS), a lysosomal storage disease. Histological lesions included vacuolation of stromal cells of the cornea, fibroblasts, chondrocytes, macrophages and renal cells. The brain was essentially normal except for moderate secondary Wallerian-type degeneration in motor and sensory tracts of the hind brain. Dermatan sulphate-uria was present and enzymology revealed negligible activity of N-acetylgalactosamine-4-sulphatase, also known as arylsulphatase B, in cultured fibroblasts and liver tissue. A novel homozygous 22 base pair (bp) deletion in exon 1 of this enzyme's gene was identified (c.103_124del), which caused aframe-shift and subsequent premature stop codon. The “Wisdom pure breed-mixed breed” test reported the dog as a cross between a Miniature and Toy Poodle.

CONCLUSIONS: The clinicopathological features are similar to those of MPS type VI as previously described in dogs, cats and other species, and this clinical diagnosis was confirmed by enzymology and molecular genetic studies. This is an autosomal recessively inherited lysosomal storage disease.

CLINICAL RELEVANCE: The prevalence of MPS VI in Miniature or Toy Poodles in New Zealand and elsewhere is currently unknown. Due to the congenital nature of the disorder, malformed pups may be subject to euthanasia without investigation and the potential genetic problem in the breed may not be fully recognised. The establishment of a molecular genetic test now permits screening for this mutation as a basis to an informed breeding policy.     

Musculoskeletal responses of 2-year-old Thoroughbred horses to early training. 3. In vivo ultrasonographic assessment of the cross-sectional area and echogenicity of the superficial digital flexor tendon

AIM: To determine if the superficial digital flexor tendon (SDFT) of young Thoroughbred horses changed in size and echogenicity in association with early race training.

METHODS: Cross-sectional area (CSA) and echogenicity were determined ultrasonographically at five levels of the SDFT of the forelimbs of 2-year-old fillies (n=14), corresponding to 4, 8, 12, 16 and 20 cm distal to the accessory carpal bone (DACB). Measurements were made before and after a 13-week period in which a trained group of seven horses was compared with another group of seven untrained horses.

RESULTS: Level below the accessory carpal bone had a significant effect on CSA and Level 8 was smaller than all other levels except Level 12, while Level 12 was smaller than Levels 4 and 20 but not different from Levels 8 and 16. There was a significant interaction between level and time due to effects observed at Level 8. The CSA at Level 8 measured pre-training was different from that of Levels 4 and 20 in both pre- and post-training groups (p<0.05), but when measured post-training was not different from any other measurement. There was also a significant interaction between treatment group and time. There was no difference between CSA for the untrained and trained groups at the pre-training observation (p=0.9), but post-training the CSA (pooled over all levels) in trained horses was significantly larger than that of the untrained horses both post-training (p=0.019) and pre-training (p=0.034), and was not different from the pretraining CSA recorded in the trained group (p=0.29). Treatment group had no effect on echogenicity (p=0.43), while echogenicity was less at the end of the trial in both trained and untrained horses (p<0.001).

CONCLUSIONS: Early training for racing was associated with an increase in mean CSA of the SDFT. Other factors such as age and maturity may play a role in limiting this increase.     

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.