Assessing the welfare of genetically altered mice.

In 2003, under the auspices of the main UK funders of biological and biomedical research, a working group was established with a remit to review potential welfare issues for genetically altered (GA) mice, to summarize current practice, and to recommend contemporary best practice for welfare assessments. The working group has produced a report which makes practical recommendations for GA mouse welfare assessment and dissemination of welfare information between establishments using a 'mouse passport'. The report can be found at www.nc3rs.org.uk/GAmice and www.lal.org.uk/gaa and includes templates for the recommended welfare assessment scheme and the mouse passport. An overview is provided below.

Control of variability.

Genetic variability is an important component of the phenotypic variation within populations; however, there are often many other contributing factors, which receive little attention. Randomization techniques or grouping factors known to contribute to variability can do much to isolate, or at least accommodate, this variability. The European Centre for the Validation of Alternative Methods addressed some of these approaches in 1998, and others are considered in this article. However, laboratory animals are living beings that respond to scientific procedures, or indeed to subtle variations in husbandry conditions, which our senses do not equip us to comprehend readily. These variations may either bias our experimental results or introduce sufficient background noise to mask differences arising from the scientific procedures. In planning an experiment, it is important to devote adequate time considering such factors and developing appropriate strategies to handle them.

Crystal structure of Escherichia coli malate synthase G complexed with magnesium and glyoxylate at 2.0 A resolution: mechanistic implications.

The crystal structure of selenomethionine-substituted malate synthase G, an 81 kDa monomeric enzyme from Escherichia coli has been determined by MAD phasing, model building, and crystallographic refinement to a resolution of 2.0 A. The crystallographic R factor is 0.177 for 49 242 reflections observed at the incident wavelength of 1.008 A, and the model stereochemistry is satisfactory. The basic fold of the enzyme is that of a beta8/alpha8 (TIM) barrel. The barrel is centrally located, with an N-terminal alpha-helical domain flanking one side. An inserted beta-sheet domain folds against the opposite side of the barrel, and an alpha-helical C-terminal domain forms a plug which caps the active site. Malate synthase catalyzes the condensation of glyoxylate and acetyl-coenzyme A and hydrolysis of the intermediate to yield malate and coenzyme A, requiring Mg(2+). The structure reveals an enzyme-substrate complex with glyoxylate and Mg(2+) which coordinates the aldehyde and carboxylate functions of the substrate. Two strictly conserved residues, Asp631 and Arg338, are proposed to provide concerted acid-base chemistry for the generation of the enol(ate) intermediate of acetyl-coenzyme A, while main-chain hydrogen bonds and bound Mg(2+) polarize glyoxylate in preparation for nucleophilic attack. The catalytic strategy of malate synthase appears to be essentially the same as that of citrate synthase, with the electrophile activated for nucleophilic attack by nearby positive charges and hydrogen bonds, while concerted acid-base catalysis accomplishes the abstraction of a proton from the methyl group of acetyl-coenzyme A. An active site aspartate is, however, the only common feature of these two enzymes, and the active sites of these enzymes are produced by quite different protein folds. Interesting similarities in the overall folds and modes of substrate recognition are discussed in comparisons of malate synthase with pyruvate kinase and pyruvate phosphate dikinase.

Glyceryl trinitrate enhances nitric oxide mediated perfusion within the equine hoof.

Laminitis, a microvascular disease of the equine hoof leads to severe lameness. Exogenous iv 1-arginine and transdermal nitric oxide donors, such as GTN, applied to the pasterns improve lameness during acute laminitis. Near Infrared spectroscopy in an earlier study showed haemostasis and ischaemia in the hoof during acute laminitis, both were alleviated by 1-arginine. Quantitative NIRS in the present study shows that transdermal GTN increases blood flow in the equine hoof. It is concluded that glyceryl trinitrate enhances nitric oxide mediated perfusion within the equine hoof in normal and chronically laminitic horses and ponies.

Nitric oxide donors as treatment for grass induced acute laminitis in ponies.

The potential for participation of the arginine-nitric oxide system in the aetiology of acute equine laminitis has been assessed. Nitric oxide (NO), produced by the action of NO synthase (NOS) on its substrate l-arginine, relaxes vascular smooth muscle to cause vasodilation. An attenuated normal vasodilatory tone may characterise the pathogenesis of acute equine laminitis. An intravenous infusion of 10% l-arginine in 0.9% saline caused vasodilatation in the hoof of a normal pony and immediate reperfusion of laminal tissues in an acutely laminitic pony, detected noninvasively by near infrared spectroscopy (NIRS), but the amino acid had little effect on systemic blood pressure. Treatment of acute laminitis with glyceryl trinitrate applied topically to the pasterns reduced the typical 'bounding pulses' in treated limbs, reduced lameness and lowered systemic blood pressure. Nitric oxide is likely to participate in the multifactorial pathogenesis of equine laminitis.

Near infrared spectroscopy of pedal haemodynamics and oxygenation in normal and laminitic horses.

The present study applies near infrared spectroscopy (NIRS) to the haemodynamics of the pedal circulation in normal and laminitic horses. NIRS is a noninvasive technique which uses changes in light absorption at 4 wavelengths to provide information on the changes in cytochrome aa3 (cyt aa3) reduction-oxidation (redox) status, and changes in the tissue concentration of oxyhaemoglobin (O2Hb), deoxyhaemoglobin (HHb) and therefore total haemoglobin (tHb). Other studies have shown NIRS to be sensitive to changes in tissue oxygenation and perfusion in human cerebral and limb circulation. In this study, the NIRS sensor was applied to the dorsal surface of horses' hooves. Normal and laminitic animals (acute and chronic) were subjected to manoeuvres (cuff tourniquet; digital vessel occlusion at the palmar surface of the pastern; lifting of contralateral limb) predicted to change pedal haemodynamics. The procedures produced changes in pedal haemodynamics and oxygenation, which were similar to those observed in the ischaemic/reperfused human forearm. Laminitic differed from normal horses: return of HHb to baseline was slower and the change in cyt aa3 more rapid than normal in cases of chronic laminitis, taken to indicate reduced O2 stores as a result of compromised basal perfusion. In acute laminitis, baseline values did not fluctuate following any of the manoeuvres, suggesting haemostasis in the diseased hoof. NIRS is a useful noninvasive method to assess pedal vascular function in normal and laminitic horses.

Health risks of housing small psittacines in galvanized wire mesh cages.

Each week over a 6-week period, 80 adult cockatiels (Nymphicus hollandicus) of either gender were dosed orally with fine particles of pure zinc or galvanized coating removed from welded wire mesh. At dosage of 32 mg/wk, all birds became severely ill and either died or were euthanatized within 2 weeks. Dosage of 2 mg/wk induced chronic illness marked by dullness, weight loss, and intermittent excretion of greenish droppings. Necropsy findings were unremarkable, except for signs suggestive of impaired gastrointestinal tract motility and histologic degenerative changes associated with focal mononuclear infiltration in the liver, kidneys, and pancreas. Tissue, especially pancreatic, contents of zinc were markedly high. Pure zinc was as toxic as galvanizing zinc. White rust, an oxidation product, also was toxic. The galvanized coating on cages and flights must be carefully wirebrushed and examined before housing psittacine birds.

Clinical and radiographical diagnosis of coenurosis cerebralis in sheep and its surgical treatment.

During the period between November 1982 and April 1984, 11 sheep showing clinical signs of infection by Coenurus cerebralis were subjected to clinical and radiographical examinations, and subsequently treated surgically for removal of the cyst. A detailed account is given of the investigations carried out and of the surgical procedure adopted for the removal of the cyst together with cystographical and surgicopathological findings. There was a successful clinical response to the removal of the cyst.

Neural and chemical control of abomasal secretion in sheep.

Abomasal secretion has been collected from Grosskopftype pouches prepared in five adult Karrakul ewes. Feeding caused a marked increase in volume, acidity and pepsin activity, whereas these factors were reduced by starvation. Both insulin-induced hypoglycaemia and electrical stimulation of the vagal nerve supply to the pouch increased the volume, total acid and pepsin secretion. It is concluded that vagal activity is an important factor in the control of abomasal secretion in sheep.

A comparative study of ketone body metabolism between the camel (Camelus dromedarius) and the sheep (Ovis aries).

1. Plasma levels of beta-hydroxybutyrate (BHB), and acetoacetate (AcAc) have been measured in camels (Camelus dromedarius) and sheep (Ovis aries). The activity of beta-hydroxybutyrate dehydrogenase (BHB-deH2) (E.C. 1.1.1.30) was studied in the rumen epithelium and the liver of these animals. 2. Concentrations of plasma BHB and AcAc in the camel were in respective order 33 and 4 times lower than that of the sheep. The ratios of BHB to AcAc were 0.61 and 4.8 for the camel and sheep, respectively. 3. The activity of BHB-deH2 in the rumen epithelium of the camel and sheep were 7.15 and 66 mumol/hr/g wet wt tissue, respectively. The activity in both species was higher in the rumen epithelium than in the liver.

The effect of thiopental sodium, methoxyflurane and halothane on the acid-base status in sheep.

Experiments have been carried out on 20 adult fat-tailed ewes to determine the effects of thiopental sodium, methoxyflurane and halothane on acid-base status of the saliva loss during prolonged surgical anaesthesia. The rate of loss of base in saliva depends on the volume of saliva produced, which fell sharply at the onset of anesthesia with the volatile anaesthesia. Plasma pH and plasma pvCO2 excess were both increased by the volatile anaesthetics but fell sharply during thiopental anaesthesia. Plasma pH and plasma PvCO2 showed no consistent relationship.

Endogenous uric acid and urea metabolism in the chicken.

1. Four chickens were used in nine successive experiments using a single injection radioisotope dilution technique to study the kinetics of endogenous uric acid and urea metabolism. 2. Starvation lowered uric acid and urea entry rates, but elevated the extent of degradation of these compounds. 3. Urea turnover time and the extent of urea pool degradation were higher, and urea excretion rate was lower than that of uric acid. 4. In colostomised chickens, the extent of uric acid and urea degradation were lower than in non-colostomised birds. 5. The average uric acid and urea entry rates in chickens fed on a diet containing 200 g protein/kg were 7.32 and 2.6 mumol/h g liver, respectively. 6. It is concluded that the contribution of uric acid and urea to the nitrogen economy of the birds is negligible.

The effect of changes in blood glucose on the motility of the duodenum in sheep.

Experiments have been carried out on four adult ewes to determine the effects on duodenal motility of intravenous or intraduodenal infusion of glucose solution and intravenous injection of insulin. At blood glucose levels below 80 mg%, the mean frequency of duodenal contractions was 1.6/min, but following the establishment of hyperglycemia by either intraduodenal or intravenous glucose infusion, contraction frequency fell to 0.9/min. The strength of contraction was unaffected.

Localization of the medullary respiratory neurons in rats by microelectrode recording.

Neuronal activity has been recorded extracellularly from the medulla of anesthetized rats. Units whose discharge frequency varied in phase with respiratory airflow were located bilaterally between 1.5 and 2 mm lateral to midline, extending from 1 mm caudal to 1.5 mm rostral to the obex, in the ventral two-thirds of the medulla. Expiratory units predominated and were intermingled with inspiratory units. Ten different patterns of discharge were distinguished, varying from a short burst at the beginning of expiration to a resting discharge which increased in frequency during either inspiration or expiration. Evidence was also obtained that fiber tracts from other areas of the brain cross midline just caudally to the obex and pass to the respiratory centers on which they apparently exert and excitatory action.

Water balance of the hen during egg formation.

Laying hens consume more water on days when an egg is laid, than on non-laying days; the daily water intake is more than double--from 115 g. to 255 g. and the number of drinks taken increases proportionately. The weight of water in the egg produced is only 32 g. The requirement for this excess of water implies a considerable metabolic stress associated with egg formation. Studies have been carried out to determine why this water is consumed, and its metabolic fate. Water ingestion started to increase about 12 h. before oviposition; it rose steadily until about 2 h. before lay, then fell sharply. Urine minute volume showed similar fluctuations. At the time when the urine output fell just before lay, plasma osmolarity was higher than usual; this might be predicted from the drop in water consumption. Alterations in the water content of the oviduct were not sufficient to explain the increased fluid intake, and total body weight remained constant in spite of the consumption of the additional 140 g. water. These findings suggest that the water has a metabolic role, as it is not retained as a net fluid surplus.

Occurrence of infection by the cestode Grillotia in Persian Gulf fish.

A cystic condition of the peritoneum of tuna fish (Thunnus thynnus) caught from the Persian Gulf is described. Parasitologic examination established widespread infection by the cestode Grillotia. An account is given of the taxonomic position of this genus.