Blood sampling and intravenous injections in Japanese quail (Coturnix coturnix japonica).

The suitability of five different sites (brachial, jugular, caudal tibial vein, external dorsal thoracic vein and the heart) were examined for blood sampling and intravenous injections in Japanese quail. In terms of safety, ease of collection, repeated collections or injections, the jugular vein was found to be the most suitable.     

Isolation and characterization of eight microsatellite loci for the study of gene flow between Testudo marginata and Testudo weissingeri (Testudines: Testudinidae)

The taxonomic status of the Greek Testudo species, marginated tortoise (Testudo marginata Schoepff, 1793) and dwarf marginated tortoise (Testudo weissingeri Bour, 1995), is controversed. In order to study the gene flow among these Greek Testudo species, eight polymorphic microsatellite markers were characterized and screened for 32 individuals. The number of alleles observed per locus ranged from two to 19. Observed heterozygosities varied from 0.235 to 0.830. These microsatellite loci were also successfully tested on seven closely related species of Testudinidae. This set of microsatellites offers an efficient tool to investigate genetic differentiation among testudinid species.     

Simple method for culture of peripheral blood lymphocytes of Testudinidae

We developed and optimized a simple, efficient and inexpensive method for in vitro culture of peripheral blood lymphocytes from the Brazilian tortoise Chelonoidis carbonaria (Testudinidae), testing various parameters, including culture medium, mitogen concentration, mitotic index, culture volume, incubation time, and mitotic arrest. Peripheral blood samples were obtained from the costal vein of four couples. The conditions that gave a good mitotic index were lymphocytes cultured at 37°C in minimum essential medium (7.5 mL), with phytohemagglutinin as a mitogen (0.375 mL), plus streptomycin/penicillin (0.1 mL), and an incubation period of 72 h. Mitotic arrest was induced by 2-h exposure to colchicine (0.1 mL), 70 h after establishing the culture. After mitotic arrest, the cells were hypotonized with 0.075 M KCl for 2 h and fixed with methanol/acetic acid (3:1). The non-banded mitotic chromosomes were visualized by Giemsa staining. The diploid chromosome number of C. carbonaria was found to be 52 in females and males, and sex chromosomes were not observed. We were able to culture peripheral blood lymphocytes of a Brazilian tortoise in vitro, for the preparation of mitotic chromosomes.     

Standard sampling techniques underestimate prevalence of avian hematozoa in willow ptarmigan (Lagopus lagopus)

A total of 68 willow ptarmigan (Lagopus lagopus L.) was collected during September 1995 from two localities in Troms County, northern Norway. Thin blood smears were prepared and examined for blood parasites. Of the 68 willow ptarmigan examined, 94% harbored one or more species of hematozoa. There were four (6%), 44 (65%), 16 (24%), and four (6%) birds infected by zero, one, two, and three species of parasites, respectively. Prevalences at the coastal locality, Kattfjord (n = 43), were Leucocytozoon lovati 86%, Trypanosoma avium (26%), and microfilariae (30%). At the inland locality, Iselvdalen (n = 25), prevalences were L. lovati 96%, T. avium 12%, and microfilariae 0%. We also searched connective tissues for the filaroid nematode Splendidofilaria papillocerca; in Kattfjord this parasite only occurred in adult hosts where prevalence was 94%, but the parasite was not found in Iselvdalen. To estimate the efficiency of parasite detection by standard blood sampling techniques, we sampled peripheral blood from the brachial wing vein and blood from the pulmonary system from willow ptarmigan. Sampling peripheral blood from the brachial vein led to underestimates of the prevalence of microfilariae. There was no significant difference between L. lovati and T. avium prevalence in blood collected from the brachial vein or deep circulation. Age of host had a strong impact on prevalence, especially for S. papillocerca and microfilariae.     

Oxidative stress, nitric oxide, endothelial dysfunction and tinnitus

To assess whether pathogenic endothelial dysfunction is involved in acute idiopathic tinnitus we enrolled 44 patients and 25 healthy volunteers. In blood from the internal jugular vein and brachial vein we determined malonaldehyde, 4-hydroxynonenal, myeloperoxidase, glutathione peroxidase, nitric oxide, L-arginine and L-ornitine, thrombomodulin (TM) and von Willebrand factor (vWF) activity during tinnitus and asymptomatic period. Higher plasma concentrations of oxidative markers and L-arginine, and lower nitric oxide and L-ornitine levels were observed in jugular blood of patients with tinnitus, there being a significant difference between brachial and jugular veins. TM and vWF activity were significantly higher in patients' jugular blood than in brachial blood. Our results suggest oxidant, TM, vWF activity production are increased and nitric oxide production reduced in brain circulation reflux blood of patients with acute tinnitus. These conditions are able to cause a general cerebro-vascular endothelial dysfunction, which in turn induce a dysfunction of microcirculation in the inner ear.     

Effect of serial blood sampling on interpretation of estradiol-17 beta involvement in laying hen calcium metabolism

Effects of long-term, low levels of exogenous estradiol-17 beta and dietary calcium on calcium metabolism and laying hen performance, and effects of serial blood-sampling, were studied. Significant 3-way (P less than 0.01) and 2-way (P less than 0.05) interactions involving sampling obscured possible main effects (Exp. 1); no main effects were found in absence of blood sampling (Exp. 2). Estradiol-17 beta appears to increase calcium utilization with very low dietary calcium only. Relatively small brachial vein samples taken only every 3-4 weeks have substantial confounding effects on interpretation of data.     

Hematological and plasma biochemical values for captive tufted capuchin monkeys (Cebus apella)

Hematological and blood biochemical parameters are of great importance in medical and veterinary practice. Unfortunately, normal reference range intervals for hematological and serum biochemical values in the tufted capuchin monkey (Cebus apella) have seldom been reported. The study reported here is based on data from blood samples collected from 44 monkeys over an 8-year period. Male and female data are displayed separately within two age categories: juveniles and adults, and effects for sex and age are examined. Significant differences between males and females are found for erythrocytes, hemoglobin, hematocrit, and α(1) globulin. Significant differences between juveniles and adults are found for neutrophils, calcium, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, inorganic phosphorus, glucose, total protein, serum iron, and some serum protein parameters and albumin/globulin ratio. These values are compared with values we previously reported, and their importance in care and well-being of captive tufted capuchin monkeys is discussed.     

Effects of landscape features and demographic history on the genetic structure of Testudo marginata populations in the southern Peloponnese and Sardinia

Testudo marginata, the largest European land tortoise, is suffering habitat degradation and destruction. Some populations, in markedly degraded habitats, are characterized by divergent morphotypes. However, the evolutionary significance of these morphotypes is of debate. Using 11 polymorphic microsatellites, we studied: (1) marginated tortoises from Sardinia that display a divergent morphotype – this population was potentially introduced from Greece; and (2) an area in the southern Peloponnese that includes a small and degraded zone in which marginated tortoises are dwarf. Genetic analyses run without any a priori assignment clearly acknowledge the specimens sampled in the territory of the dwarf form as a single group whilst Sardinian specimens are clustered with other specimens from the northern part of the area sampled in Greece. Demographic analyses suggest that Sardinian tortoises originated recently from some of the populations sampled in the northern part of the area sampled in Greece. Over locations sampled in Greece, a landscape‐genetic analysis allowed us to detect potential landscape features that may reduce gene flow between the dwarf form territory and surrounding areas. Our results suggest that the territory of the dwarf form is particularly propitious for marginated tortoises and that conservation regulations in Greece should be reinforced to protect this area from increasing impact of human activities changing from traditional agriculture to mechanization and extensive use of chemicals. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 591–606.     

Distinct signalling pathways regulate sprouting angiogenesis from the dorsal aorta and the axial vein

Angiogenesis, the formation of new blood vessels from pre-existing vessels, is critical to most physiological processes and many pathological conditions. During zebrafish development, angiogenesis expands the axial vessels into a complex vascular network that is necessary for efficient oxygen delivery. Although the dorsal aorta and the axial vein are spatially juxtaposed, the initial angiogenic sprouts from these vessels extend in opposite directions, indicating that distinct cues may regulate angiogenesis of the axial vessels. We found that angiogenic sprouts from the dorsal aorta are dependent on vascular endothelial growth factor A (Vegf-A) signalling, and do not respond to bone morphogenetic protein (Bmp) signals. In contrast, sprouts from the axial vein are regulated by Bmp signalling independently of Vegf-A signals, indicating that Bmp is a vein-specific angiogenic cue during early vascular development. Our results support a paradigm whereby different signals regulate distinct programmes of sprouting angiogenesis from the axial vein and dorsal aorta, and indicate that signalling heterogeneity contributes to the complexity of vascular networks.     

Oxidative stress,nitric oxide,endothelial dysfunction and tinnitus

To assess whether pathogenic endothelial dysfunction is involved in acute idiopathic tinnitus we enrolled 44 patients and 25 healthy volunteers. In blood from the internal jugular vein and brachial vein we determined malonaldehyde, 4-hydroxynonenal, mieloperoxidase, glutathione peroxidase, nitric oxide, l-arginine and l-ornitine, thrombomodulin (TM) and von Willebrand factor (vWF) activity during tinnitus and asymptomatic period.

Higher plasma concentrations of oxidative markers and l-arginine, and lower nitric oxide and l-ornitine levels were observed in jugular blood of patients with tinnitus, there being a significant difference between brachial and jugular veins. TM and vWF activity were significantly higher in patients' jugular blood than in brachial blood.

Our results suggest oxidant, TM, vWF activity production are increased and nitric oxide production reduced in brain circulation reflux blood of patients with acute tinnitus. These conditions are able to cause a general cerebro-vascular endothelial dysfunction, which in turn induce a dysfunction of microcirculation in the inner ear.     

Blood sample collection in lizards

Blood sample collection in lizards is an important technique. This column describes techniques for sample collection from the ventral coccygeal vein and the ventral abdominal vein.     

Synchronization of estrus in early diestral dairy heifers with Prostaglandin F(2)alpha and estradiol benzoate

Following observation of estrus, 134 Holstein heifers were given injections of Prostaglandin F(2)alpha (PGF(2)alpha) between Days 5 and 10 of their cycle (estrus = Day 0). They were then randomly assigned to either a group receiving 400 mug of estradiol benzoate (E(2)B) 40 h or maintained as controls. Heifers observed in estrus within 120 h of PGF(2)alpha administration were inseminated (approximately 12 h after initial observation of estrus). Blood samples for progesterone determination were drawn from the coccygeal vein on Days 15 and 21 after insemination. Pregnancy was confirmed by palpation per rectum between Days 5.0 and 60 post insemination. When control and treated heifers were compared it was found that a higher percentage of heifers treated with E(2)B exhibited estrus after PGF(2)alpha, but there had been no effect on subsequent progesterone concentrations or pregnancy rates.     

Protective Effect of a Fermented Substance from Saccharomyces cerevisiae on Liver Injury in Mice Caused by Acetaminophen

The protective effect of a fermented substance from Saccharomyces cerevisiae (FSSC) on liver injury caused by acetaminophen (AAP) was studied in mice. Mice were pretreated with FSSC (0.5–2.0 g/kg, p.o.) for 4 d, and on the fourth day, the mice received an overdose of AAP (500 mg/kg, i.p.). Subsequently, they were sacrificed at 7 h, and blood was drawn from the abdominal vein and liver samples were collected. Histological and biochemical examinations revealed that the administration of AAP caused liver injury in the mice, including increases in plasma alanine aminotransferase and asparate aminotransferase activities and decreases in the hepatic reduced form of glutathione (GSH) content and antioxidant enzyme activities. Prior to AAP treatment, the mice pretreated with FSSC showed significantly reduced levels of alanine aminotransferase (ALT) and aspirate aminotransferase (AST) activity. Liver histology in the FSSC-pretreated mice was significant. In these mice, pretreatment with FSSC also served to reduce hepatic GSH depletion and the inhibition of antioxidant enzyme activity caused by AAP overdose. In conclusion, oral administration of FSSC significantly reduced AAP-induced hepatic injury in the mice.     

Protective effect of a fermented substance from Saccharomyces cerevisiae on liver injury in mice caused by acetaminophen

The protective effect of a fermented substance from Saccharomyces cerevisiae (FSSC) on liver injury caused by acetaminophen (AAP) was studied in mice. Mice were pretreated with FSSC (0.5-2.0 g/kg, p.o.) for 4 d, and on the fourth day, the mice received an overdose of AAP (500 mg/kg, i.p.). Subsequently, they were sacrificed at 7 h, and blood was drawn from the abdominal vein and liver samples were collected. Histological and biochemical examinations revealed that the administration of AAP caused liver injury in the mice, including increases in plasma alanine aminotransferase and asparate aminotransferase activities and decreases in the hepatic reduced form of glutathione (GSH) content and antioxidant enzyme activities. Prior to AAP treatment, the mice pretreated with FSSC showed significantly reduced levels of alanine aminotransferase (ALT) and aspirate aminotransferase (AST) activity. Liver histology in the FSSC-pretreated mice was significant. In these mice, pretreatment with FSSC also served to reduce hepatic GSH depletion and the inhibition of antioxidant enzyme activity caused by AAP overdose. In conclusion, oral administration of FSSC significantly reduced AAP-induced hepatic injury in the mice.     

Serum biochemical values in fallow deer (<Emphasis Type="Italic">Dama dama</Emphasis> L.) from different habitats in Croatia

Serum biochemical values were determined in blood samples collected from 48 shot fallow deer from the Brijuni islands and 45 sedated fallow deer (Dama dama L.) from hunting grounds in the continental part of Croatia. The parameters were compared with regard to age, sex and habitat. Statistically significant differences were found for serum total protein concentration, alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase activity between young and adult island deer, as well as for total protein, triacylglyceride, cholesterol concentration and alkaline phosphatase activity between young and adult continental deer. In young animals, island males had higher albumin concentrations, while continental males had higher alkaline phosphatase values than females. In adult animals, island males had a higher blood urea nitrogen concentration, while continental males had higher albumin and cholesterol concentrations, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and creatine kinase activities. In this group, males exhibited lower bilirubin and triacylglyceride concentrations than females. Our results indicate that besides age, sex and sampling method, nutritional and environmental factors should be considered when evaluating serum biochemical parameters of fallow deer     

Intestinal absorption and plasma transport of lipids in chicks and rats

Labelled oleic acid was introduced into the duodenum of chicks in which the pancreatic-duodenal vein and brachial artery had been cannulated, and blood samples were withdrawn. Similar experiments were performed in rats which had not been venously cannulated. In rat plasma, over 92% of the label was found in the triglycerides, whereas in the chick 64% of the label was in the triglycerides and 27-31% in the free fatty acids. In the rat, over 85% of the lipid label circulating in the plasma was found in lipoproteins with hydrated density less than 1.006, whereas in the chick only 25% of the label was in this fraction and the majority of label was found with density greater than 1.063. It thus appears that both release and transport of fatty acids from the intestinal mucosa in chicks differs from the rat.     

Serum cortisol and haematological,biochemical and antioxidant enzyme variables in horse blood sampled in a slaughterhouse lairage,immediately before stunning and during exsanguination

The aim of the study was to determine changes of serum cortisol and biochemical, haematological and antioxidant enzyme variables in the blood of horses sampled during the pre-slaughter period (in the lairage and in the stunning box) and during exsanguination. A total of 24 Slovenian warm-blooded horses were observed. Blood samples for determination of serum cortisol and biochemical, haematological (red blood cell count, haematocrit, haemaglobin concentration) and antioxidant enzyme (whole blood superoxide dismutase and glutathione peroxidase) variables were collected by venipuncture of the left jugular vein in the lairage pen, 60 min before stunning (lairage) and immediately before stunning (stunning box). At exsanguination, blood samples were collected from the wound at the time of jugular vein sticking. During blood collection in the lairage pen and in the stunning box, horses were gently restrained with a halter. They were stunned using a penetrating captive bolt pistol impelled by air and were bled by jugular vein sticking. Horses were physically active in the lairage pen and in the race before entering the stunning box. After stunning, the horses showed paddling movements with their legs. In horses, the plasma lactate and glucose concentrations, the serum potassium concentration, the activities of the serum muscle enzymes aspartate aminotransferase and creatine kinase, and values of most of the other biochemical (Table 1) and haematological variables (Table 2), were significantly (P < 0.05) higher at exsanguination, than in blood sampled while they were in the lairage and in the stunning box. The serum concentrations of cortisol and chloride and the activities of alanine aminotransferase and antioxidant enzymes were not significantly different between the pre-slaughter period and exsanguination. All selected blood variables were not significantly different between the lairge and the stunning box sampling time, indicating no physiological stress responses of the investigated horses to stressors, such as novelty of the pre-slaughter environment and handling, present in the slaughterhouse between the lairage and the stunning box. However, the significantly higher values, at exsanguination, for the plasma lactate and glucose concentrations, serum muscle enzyme activities and haematological variables, than during the pre-slaughter period, might partially be attributed to stimulation of the sympathetic nervous system, caused by stunning and bleeding.     

Sex differences in DHEA and estradiol during development in a wild songbird: Jugular versus brachial plasma

Sexual differentiation of the brain has traditionally been thought to be driven by gonadal hormones, particularly testosterone (T). Recent studies in songbirds and other species have indicated that non-gonadal sex steroids may also be important. For example, dehydroepiandrosterone (DHEA) - a sex steroid precursor that can be synthesized in the adrenal glands and/or brain - can be converted into active sex steroids, such as 17β-estradiol (E₂), within the brain. Here, we examine plasma DHEA and E₂ levels in wild developing European starlings (Sturnus vulgaris), from hatch (P0) to fledging (P20). Blood samples were collected from either the brachial vein (n = 143) or the jugular vein (n = 129). In songbirds, jugular plasma is enriched with neurally-synthesized steroids and, therefore, jugular plasma is an indirect measure of the neural steroidal milieu. Interestingly, brachial DHEA levels were higher in males than females at P4. In contrast, jugular DHEA levels were higher in females than males at P0 and P10. Brachial E₂ levels were higher in males than females at P6. Surprisingly, jugular E₂ levels were not high and showed no sex differences. Also, we calculated the difference between brachial and jugular steroid levels. At several ages, jugular steroid levels were lower than brachial levels, particularly in males, suggesting greater neural metabolism of circulating DHEA and E₂ in males than females. At a few ages, jugular steroid levels were higher than brachial levels, suggesting neural secretion of DHEA or E₂ into the general circulation. Taken together, these data suggest that DHEA may play a role in brain sexual differentiation in songbirds.     

Distribution of some enzymes associated with the metabolism of glutamate, aspartate, gamma-aminobutyrate and glutamine in cat spinal cord

The activities of glutamine synthetase, glutaminase, glutamate decarboxylase, GABA aminotransferase, glutamate dehydrogenase, and aspartate aminotransferase were measured in four areas of the cat spinal cord and in dorsal and ventral roots. Five of the six enzymes showed identical distribution patterns; i.e. the activities in the dorsal and ventral gray matter were equal and those of dorsal and ventral white matter were equal. No statistical differences in the mean enzyme activities in the dorsal and ventral roots were found. Glutamate decarboxylase was the only enzyme which had a different pattern. The enzyme activity in dorsal gray was twice that of ventral gray; the same pattern as the GABA concentration in both these areas. The glutamine synthetase activities in the cord areas and roots correlated with the glutamine distribution reported earlier. Thus, the distribution of glutamine (not a transmitter) and GABA (questionable transmitter) in gray matter are dictated by their synthesizing enzymes, whereas the distribution of glutamate and aspartate (likely transmitter suspects) cannot be explained on the basis of enzyme activities. Therefore, the enzyme activities may be related to the amino acid levels primarily in metabolic compartments, whereas the excess of certain amino acids in specific areas of the cord and roots may be related to functional compartments accumulated for use in synaptic transmission.     

一种动态检测小鼠微量血中T细胞受体重排删除环含量的方法

目的:通过检测比较外周血(颈动脉大量采血和微量隐静脉采血)、胸腺组织和脾脏组织等不同成分或组织中T细胞受体重排删除环(T cell receptor rearrangement excision circles,TREC)的含量,并建立一种通过微量外周血PCR预扩增的方法,评估胸腺输出功能。方法:采用C57BL/6小鼠作为实验对象,分成幼年组(5周龄,n=10)和成年组(15周龄,n=10)。经过隐静脉采血及颈动脉采血后处死小鼠,取小鼠胸腺器官和脾脏器官,提取基因组DNA(g DNA),隐静脉微量血g DNA通过PCR预扩增,提纯后再和颈动脉血、胸腺组织和脾脏组织g DNA一起进行实时定量PCR,分析各组织成分TREC的表达水平及差别。结果:幼年组胸腺组织及外周血中TREC的含量比成年组高,且二者趋势基本一致;而脾脏组织结果与其相反;幼年组小鼠胸腺组织中TREC的含量比脾脏组织中高,成年组小鼠结果与之相反;微量外周血经过预扩增后再进行实时定量PCR的结果与直接定量PCR结果一致,且更高效。结论:研究提供了一种新型高效的动态检测T细胞受体重排删除环和检测胸腺输出功能的方法。