Susceptibility to onion-induced hemolysis in dogs with hereditary high erythrocyte reduced glutathione and potassium concentrations.

The hemolytic effect of onion consumption in dogs with hereditary high erythrocyte reduced glutathione and potassium concentrations (designated HK dogs) was compared with that in clinically normal dogs. Twelve hours after oral administration of boiled onions (200 g/dog), hemoglobin concentration decreased to 84.4% of the initial value in HK dogs; it decreased only to 90.5% in clinically normal dogs. At 24 hours, methemoglobin concentration was significantly (P less than 0.05) higher in HK dogs than in clinically normal dogs. The concentration of erythrocyte oxidized glutathione increased about fivefold at 10 hours in HK dogs, whereas it did not change during the experimental period in clinically normal dogs. In addition, at 12 hours, the proportion of erythrocytes containing Heinz bodies increased to 24.4% in HK dogs, but increased only to 1.2% in clinically normal dogs. Thus, results indicated that HK dogs were more susceptible to the oxidant action of onions than were clinically normal dogs.     

Laboratory diagnosis of canine GM2-gangliosidosis using blood and cerebrospinal fluid.

In the present study, laboratory techniques were used to diagnose canine GM2-gangliosidosis using blood and cerebrospinal fluid (CSF) that can be collected noninvasively from living individuals. Lysosomal acid beta-hexosaminidase (Hex) was measured spectrofluorometrically using 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide and 4-methylumbelliferyl 7-(6-sulfo-2-acetamido-2-deoxy-beta-D-glucopyranoside) as substrates. Main isoenzymes A and B of Hex in leukocytes were also analyzed using cellulose acetate membrane electrophoresis. GM2-ganglioside in CSF was detected and determined quantitatively by using thin-layer chromatography/enzyme-immunostaining method with anti-GM2-ganglioside antibody. In normal dogs, Hex activities could be determined in leukocytes, serum, and CSF and the total activities were markedly reduced in all the enzyme sources in a dog with Sandhoff disease. Electrophoresis of a leukocyte lysate from a normal dog showed that the Hex A and Hex B were not separated distinctively with formation of a broad band, whereas there were no bands in electrophoresis of a lysate from a dog with Sandhoff disease, showing a deficiency in the total enzyme activity. GM2-ganglioside could be detected and determined quantitatively in as little as 100 microl of canine CSE GM2-ganglioside in CSF in a dog with Sandhoff disease increased to 46 times the normal level. In conclusion, the methods in the present study are useful for diagnosis of canine GM2-gangliosidosis. These techniques enable definitive and early diagnosis of canine GM2-gangliosidosis even if tissues and organs cannot be obtained.     

Oral administration of L-citrulline changes the concentrations of plasma hormones and biochemical profile in heat-exposed broilers

We examined the effects of oral administration of L-citrulline (L-Cit) on plasma metabolic hormones and biochemical profile in broilers. Food intake, water intake, and body temperature were also analyzed. After dual oral administration (20 mmol/head/administration) of L-Cit, broilers were exposed to a high ambient temperature (HT; 30 ± 1°C) chamber for 120 min. Oral administration of L-Cit reduced (p < .001) rectal temperature in broilers. Food intake was increased (p < .05) by heat stress, but it was reduced (p < .05) by L-Cit. Plasma levels of 3,5,3′-triiodothyronine, which initially increased (p < .0001) due to heat stress, were reduced (p < .01) by oral administration of L-Cit. Plasma insulin levels were increased by heat exposure (p < .01) and oral L-Cit (p < .05). Heat stress caused a decline (p < .05) in plasma thyroxine. Plasma lactic acid (p < .05) and non-esterified fatty acids (p < .01) were increased in L-Cit-treated heat-exposed broilers. In conclusion, our results suggest that oral L-Cit can modulate plasma concentrations of major metabolic hormones and reduces food intake in broilers.     

Single nucleotide polymorphism in avian uncoupling protein gene is associated with thermoregulation in chicks

Avian uncoupling protein (av-UCP) is a key protein for thermoregulation in poultry. A single nucleotide polymorphism (SNP) in the av-UCP gene has been reported in chickens. The purpose of the current study was to clarify the association between this av-UCP gene mutation and thermoregulation in chickens. Wild and mutant type chicks for the av-UCP gene SNP (g. 1270 of the av-UCP gene exon 3 with C to T substitution and amino acid substitution) were exposed to high ambient temperature. Rectal temperature, radiation temperature on the body surface, and the expression of heat dissipation behavior (wing drooping and panting) during heat exposure were measured. In addition, oxygen consumption rate in the thermoneutral zone in wild and mutant type chicks was measured. Changes in wing temperature during heat exposure in wild-type chicks were lower than those in mutants. The latency of continuous wing drooping during heat exposure in wild-type chicks was shorter than in mutant chicks. It was also found that the SNP in the av-UCP gene caused reduced oxygen consumption. These results suggest that the av-UCP gene mutation affects thermoregulation, especially heat production, in chickens.     

Use of canine red blood cell with high concentrations of potassium, reduced glutathione, and free amino acid as host cells for in vitro cultivation of Babesia gibsoni

OBJECTIVE: To determine the usefulness of canine RBC with high concentrations of potassium, reduced glutathione (GSH), and amino acid(i.e., HK cells) for in vitro cultivation of Babesia gibsoni. ANIMALS: RBC were obtained from 3 dogs that had inherited HK cells and from 3 genetically unaffected dogs that, therefore, had RBC with lower potassium (LK) concentrations (i.e., LK cells). PROCEDURES: First, B. gibsoni were cultivated using HK or LK cells in alpha-modification of Eagle medium, consisting of Earle salts with glutamine and without ribosides, deoxyribosides, and sodium bicarbonate under a humidified atmosphere containing 5% CO2 at 37 C. Second, parasites were cultivated with LK- or HK-cell lysates. Finally, HK cells were separated into 3 fractions (bottom, middle, top layers) by density gradient centrifugation, and B. gibsoni were cultivated with each of the HK-cell fractions. In addition, the concentrations of free amino acids and reduced glutathione (GSH) in each HK-cell fraction were measured. RESULTS: B. gibsoni preferentially multiplied in HK-cell cultures rather than in LK-cell cultures. Furthermore, the addition of HK-cell lysate to the culture medium resulted in enhanced multiplication of the parasites. Higher multiplication of the parasites was observed in HK cells from the top layer, compared with HK cells from the middle and bottom layers. The HK cells from the top layer had higher concentrations of glutamate, aspartate, and GSH, compared with HK cells from the middle and bottom layer. CONCLUSIONS: Canine HK cells are useful host cells for in vitro cultivation of B. gibsoni, and the high concentrations of glutamate, aspartate, and GSH may result in enhancement of multiplication of the parasites in HK cells.     

GM1 gangliosidosis in shiba dogs

A six-month-old shiba dog with a one-month history of progressive motor dysfunction showed clinical signs of a cerebellar disorder, including ataxia, dysmetria and intention tremor of the head. Histopathological and ultrastructural studies revealed distended neurons packed with membranous cytoplasmic bodies throughout the central nervous system. The activities of lysosomal acid beta-galactosidase in its leucocytes and liver were less than 2 per cent of the control levels, and the compound accumulated in the brain was identified as GM1 ganglioside. A sibling which died immediately after birth was shown to have a beta-galactosidase deficiency in the brain and visceral organs. A family study revealed that the sire and dam of the probands were heterozygotes with approximately half of the normal level of beta-galactosidase activity, suggesting an autosomal recessive pattern of inheritance.     

Rapid and simple mutation screening of G(M1) gangliosidosis in Shiba dogs by direct amplification of deoxyribonucleic acid from various forms of canine whole-blood specimens.

This report describes a rapid and simple method for mutation screening of G(M1) gangliosidosis in Shiba dogs by direct amplification of DNA from canine whole-blood specimens using a novel polymerase chain reaction (PCR) reagent cocktail, which can eliminate the DNA extraction process and amplify the genomic DNA directly from human or murine whole blood. The strategy of this mutation screening is based on the identification of a nucleotide deletion by restriction enzyme analysis, coupled with the direct PCR amplification. The target sequence of the canine beta-galactosidase gene could be amplified directly from various forms of canine whole-blood specimens, including anticoagulated blood, blood stored frozen for 1 year, dried blood held in filter paper for 1 year at room temperature, and dry powder of blood stripped from Giemsa-stained blood films, which had been prepared 10 years earlier, resulting in the determination of genotypes in all the specimens. This method simplified the molecular diagnosis and carrier screening of G(M1) gangliosidosis in Shiba dogs, making it simple to examine specimens from the large, widely distributed population of these dogs.     

Serum concentration and properties of alpha-fetoprotein and serum level of albumin in sucking piglets

Porcine alpha-fetoprotein (AFP) was purified from the sera of four-day-old piglets using an immunoadsorbent column. The molecular weight of the AFP was 80,000 when determined by gel filtration on Sephadex G-200 and 75,000 when determined by sodium dodecylsulphate polyacrylamide gel electrophoresis. The isoelectric point of the AFP was pH 4.85 at 0 degree C. The maximum concentration of serum AFP was reached on the fourth day after birth (mean value 1.1 mg ml-1), and it then decreased to 10 micrograms ml-1 when the piglets were 35 days old. The concentration of serum albumin increased rapidly between birth and seven days old, reaching 16 to 17 mg ml-1 at seven days old. Between birth and seven days old, the serum concentrations of AFP and albumin were approximately inversely proportional.