Tadpoles fed supplemented diet with probiotic bacterium isolated from the intestinal tract of bullfrog Lithobates catesbeianus: Haematology, cell activity and electron microscopy.

The aim of this study is to select and isolate autochthonous bacteria with probiotic potential for use in a supplemented diet for bullfrog tadpoles, Lithobates catesbeianus. A total of 20 strains of lactic acid bacteria were isolated. Nine out of these were used in the following in vitro assays: antagonism against pathogenic bacteria (ANT), antimicrobial activity from extracellular compounds (MIC), tolerance to bile salts (TBS), pH reduction, protease production, sensitivity to antimicrobial tetracycline, cell viability, growth rate and doubling time. Using these data was defined an ideotype (ideal strain) based on the best results. Distances were estimated with the Mahalanobis (D2) test, and the best candidates, presenting the shortest ideotype distances, were considered to be used. The best strain was found to be Lactobacillus plantarum because it presented 10.00 ± 0.50 mm of ANT against Aeromonas hydrophila, 3.99 ± 0.01 of MIC independent of pathogenic bacteria, 85.07 ± 0.01 of TBS, 4.20 ± 0.02 of final pH, 17.67 ± 1.15 of protease production, 13.50 ± 2.00 sensitivity to antimicrobial tetracycline, 9.36 ± 0.04 of cell viability, 0.20 ± 0.00 of growth rate and 3.46 ± 0.00 doubling time. Therefore this probiotic candidate was then supplemented (2.045 ± 1.07 × 107 colony forming unities. g-1) into the diets of bullfrog tadpoles for a period of 42 days. At the end of the trial, samples of blood and intestines were collected to verify the haematological alterations and the intestinal morphology using transmission and scanning electron microscopy. Tadpoles fed the supplemented diet showed successful lactic acid bacterium colonisation, an increased number of circulating thrombocytes, monocytes, eosinophil and LG-PAS+ and also an increase in the length and density of intestinal microvilli. This study shows the feasibility of using probiotics isolated from farmed bullfrogs as a supplement in the diets of tadpoles, providing a promising alternative for modulating the health of these animals.

Profile of cortisol, glycaemia, and blood parameters of American Bullfrog tadpoles Lithobates catesbeianus exposed to density and hypoxia stressors / Perfil de cortisol, glicemia, e parâmetros sanguineos de girinos de rã-touro Lithobates catesbeianus expostos a diferentes densidades e hipóxia

The aim of this study was to evaluate alterations to the physiological profile (cortisol, glycaemia, and blood parameters) of Lithobates catesbeianus caused by the stressors density and hypoxia. The organisms were in the prometamorphosis stage and exposed to different tadpole densities: 1 tadpole/L (T1), 5 tadpoles/L (T2), and 10 tadpoles/L (T3) for 12 days. The blood was collected through the rupture of the caudal blood vessel and collected under normoxia (immediate collection) and hypoxia (after 15 minutes of air exposure) conditions. Cortisol levels rose on the fourth and eighth days of treatment and returned to basal levels by the end of the experiment. The stressor mechanisms tested did not affect glycaemia. White blood cells (total number of lymphocytes, neutrophils, and eosinophils) showed a significant difference at the twelfth day of the experiment when compared with the start of the experiment. We concluded that, under controlled conditions, a density of up to 10 tadpoles/L and air exposure for 15 minutes did not cause harmful physiological alterations during the experimental period. The answer to these stressors maybe was in another hormonal level (corticosterone).(AU)

O objetivo deste estudo foi avaliar a resposta fisiológica (cortisol, glicemia e parâmetros sanguineos) de girinos de rã-touro (Lithobates catesbeianus) em diferentes densidades e após exposição aérea. Os animais utilizados no experimento estavam entre os estágios 31 a 39, na fase de pró-metamorfose sendo testados 1 girino/L (Tratamento 1), 5 girinos/L (Tratamento 2) e 10 girinos/L (Tratamento 3), conduzidos em 3 réplicas simultâneas durante 12 dias. O sangue foi retirado por rompimento do vaso caudal na condição de Normóxia - N (tempo zero) e Hipóxia - H (tempo de 15 minutos de exposição ao ar). Foi observado um aumento nos valores de cortisol, aos 4 e 8 dias de exposição aérea retornando aos valores basais ao final do experimento, apesar de não haver diferenças significativas. A glicemia não apresentou diferenças significativas quanto aos estressores aplicados. Os parâmetros hematológicos da série branca, principalmente, o número de linfócitos, neutrófilos e eosinófilos mostraram diferença significativa aos 12 dias de experimentação quando comparados com o momento zero; concluindo-se que, em condições controladas, o adensamento de até 10 girinos/litro e a exposição aérea por 15 minutos não apresentou danos aos girinos de rã-touro durante o período experimental. O padrão de resposta a estes estímulos talvez seja expresso em outro nível hormonal (corticosterona).(AU)

[Analysis of six phenolic environmental estrogens in bullfrog blood by using dispersive solid-phase extraction and ultrafast liquid chromatography-tandem mass spectrometry].

A rapid, sensitive and accurate method for the simultaneous determination of six phenolic environmental estrogens, i. e., bisphenol A (BPA), diethylstilbestrol (DES), dienestrol( DE), hexestrol (HEX), 4-( tert-octyl)-phenol (4-tOP) and 4-nonylphenol (4-NP) in bullfrog blood by dispersive solid-phase extraction-ultrafast liquid chromatography-tandem mass spectrometry (dSPE-UFLC-MS/MS) was established. After protein precipitation, bullfrog blood samples were cleaned-up by dSPE method with ethylenediamine-functionalized Fe3O4, magnetic polymers (EDA-MPs) as adsorbent. The effects of precipitation solvents, adsorption time and the amount of EDA-MPs used on the recoveries of six phenolic environmental estrogens were investigated in detail. Chromatographic separation was performed on a Shim-pack XR-ODS II analytical column (100 mm x 2.0 mm, 2.2 microm). The mass spectrometer was operated by using electrospray ion (ESI) source in the multiple reaction monitoring (MRM) mode. The results showed that the linearities were in the range of 0.5-100.0 microg/L with correlation coefficients (r2) not less than 0.999 6 for all the six phenolic environmental estrogens. The lim- its of quantification (LOQs) (S/N > 10) in bullfrog blood samples were between 0. 075 microg/L and 0.40 microg/L. The recoveries were between 95.0% and 110.0% at three spiked levels. The precision values expressed as relative standard deviations (RSDs) were in the range of 0.6%-6.3%. The developed method can be applied to the routine analysis of the six phenolic environmental estrogens in bullfrog blood samples.

Cytochemical and immunocytochemical characterization of blood cells and immunohistochemical analysis of spleen cells from 2 species of frog, Rana (Aquarana) catesbeiana and Xenopus laevis.

BACKGROUND: Mechanisms of amphibian diseases are not characterized as well as those in domestic mammalian species. Antemortem laboratory testing is limited in frogs, presenting a diagnostic challenge to zoos, laboratories, and exotic veterinarians. OBJECTIVE: This study aimed to characterize blood cells and splenic cells from 2 anuran species based on characteristics identified by Wright staining, cytochemical staining, and immunochemical analysis and on histologic examination of spleens. METHODS: Blood specimens and spleens were obtained from 2 species of frog, the American bullfrog (Rana [Aquarana] catesbeiana) and the African clawed frog (Xenopus laevis). Blood smears were evaluated after Wright staining and cytochemical staining for α-naphthyl butyrate esterase (NBE), chloroacetate esterase (CAE), myeloperoxidase (PER), Sudan black B (SBB), and leukocyte alkaline phosphatase (LAP) reactions and for immunoreactivity for antibodies against CD3ε, CD79a, and BLA.36 antigens. Histologic sections of spleen were evaluated after staining with H&E and for immunoreactivity for CD3ε, CD79a, and BLA.36 antigens. RESULTS: In bullfrogs, neutrophils, eosinophils, and monocytes were positive for some or all of the following: NBE, CAE, PER, and SBB; lymphocytes occasionally were positive for CAE. In clawed frogs, neutrophils, basophils, and monocytes were positive for some or all of the following: NBE, CAE, PER, and SBB; eosinophils occasionally were positive for CAE and PER, and lymphocytes were negative for all cytochemical stains. LAP was not a useful marker for any leukocyte type. In both species, peripheral blood lymphocytes were strongly immunoreactive for CD3ε, CD79a, and BLA.36. In splenic tissue, histologic patterns varied and there was diffuse immunoreactivity for CD79a and BLA.36 with focal reactivity for CD3ε, but with different distribution patterns in each species. CONCLUSION: Cytochemical and immunochemical analysis of cells may be helpful in identification and characterization of amphibian blood cells and splenic cells for evaluation of the health of these animals.

Plasma pharmacokinetics of selamectin after a single topical administration in the American bullfrog (Rana catesbeiana).

Parasitism is common in wild and captive amphibians; however, pharmacologic data are lacking for anthelmintic drugs. This study was developed to determine the plasma pharmacokinetics of selamectin after topical administration in bullfrogs. Thirty-two adult American bullfrogs (Rana catesbeiana) were randomly assigned into eight groups of four with each group representing a different collection time point. Seven groups received selamectin (6 mg/ kg) topically and the remaining group served as the untreated control group. One group of frogs was euthanized and blood samples immediately collected on days 0 (control), 1, 5, 10, 15, 20, 25, and 30. Plasma was analyzed for selamectin using high performance liquid chromatography with fluorescence detection. Individual samples were analyzed, then data were reported as the mean of the four frogs at each time point. A histologic evaluation of the lung, liver, kidney, and skin tissues was performed and none of the frogs showed histologic evidence of toxicity due to selamectin administration. The mean peak plasma concentration was 162.5 +/- 42.3 ng/ml, area under the curve was 2,856 ng day/ml, mean residence time was 12.2 days, and disappearance half-life was 1.87 days. Based on the plasma pharmacokinetics, bullfrogs appear to absorb selamectin very efficiently, concentrations reach high levels in the plasma, and there were no apparent histologic effects from single dose administration.

Identification of immunoreactive plasma and stomach ghrelin, and expression of stomach ghrelin mRNA in the bullfrog, Rana catesbeiana.

In this study, we established a radioimmunoassay (RIA) specific for ghrelin from the bullfrog Rana catesbeiana using a novel antibody raised against the C-terminal amino acid sequence of bullfrog ghrelin [13-28]. We also examined the distribution of ghrelin-producing cells in the stomachs of bullfrogs using this antibody and a cRNA probe specific for the bullfrog ghrelin gene. Ghrelin levels in plasma and stomach extracts were approximately 150 fmol/ml and 83-135 fmol/mg wet tissue, respectively. Reverse-phase high performance liquid chromatographic analysis, combined with bullfrog ghrelin RIA, revealed that ghrelin immunoreactivity in the stomach was composed of non-acylated ghrelin (des-acyl ghrelin) and several acylated forms of ghrelin bearing different fatty acid modifications, which could induce increases in intracellular Ca2+ in cells expressing the rat GH secretagogue receptor. In the stomach, the major storage form was acylated ghrelin. In bullfrog plasma, however, the majority of ghrelin immunoreactivity was des-acyl ghrelin and C-terminal fragments of frog ghrelin. Acylated ghrelin forms comprised only minor peaks. Ghrelin-immunopositive and ghrelin mRNA-expressing cells were observed within the mucosal layer of the stomach. Following starvation, significant increases in plasma ghrelin levels and stomach ghrelin mRNA levels were observed as early as 10 days after starvation. These results indicate that ghrelin is present in the stomach and plasma of the bullfrog, which can be detected with our novel antibody. Interestingly, the primary storage form of ghrelin in the stomach differed from the circulating form dominating in the plasma. Furthermore, increases in ghrelin levels in plasma and mRNA levels in the stomach after starvation suggest the possible involvement of ghrelin in energy homeostasis in the bullfrog.

Blood and urine physiological values in farm-cultured Rana catesbeiana (Anura: Ranidae) in Argentina

A total of 302 samples of healthy farm-cultured Rana catesbeiana specimens (9-21 months-old, 50- 350 g liveweight, 50% each sex) from the north-east of Argentina, were analyzed through spectrophotometry, electrophoresis, densitometry, refractometry and microscopy in order to obtain blood and urine normal values. Confidence intervals (p<0.05) for PCV (28.6-31.6%), RBC (0.40-0.44 T/L), MCV (686-732 fL), hemoglobin (6.41-7.20 g/dL), MCH (151-164 pg), MCHC (22.6-24.0%), WBC (18.7-22.3 G/L), neutrophils (58.4-63.4%), lymphocytes (23.9-29.8%), monocytes (2.1-3.8%), eosinophils (4.6-7.0%), basophils (2.9-4.1%), bleeding time (289-393s), coagulation time (452-696s), prothrombin time (76-128s), urinary density (1.0061-1.0089 g/mL), urinary pH (6,38-6.96), fibrinogen (0.59-0.99 g/dL), total protein (4.19-4.49 g/dL), albumin (1.49-1.67 g/dL), alpha-1 globulin (0.20-0.24 g/dL), alpha-2 globulin (0.48-0.54 g/dL), beta globulin (0.68-0.77 g/dL), gamma globulin (1.28-1.42 g/dL), albumin/globulin ratio (0.50-0.58), creatinine (4.09-5.56 mg/L), urea (76.1-92.4 mg/L), uric acid (11.5-15.4 mg/L), triglycerides (0.34-0.52 g/L), total cholesterol (0.56-0.67 g/L), HDL-C (0.03-0.05 g/L), LDL-C (0.34-0.44 g/L), alpha lipoprotein (6.01-8.67%), beta lipoprotein (91.3-93.9%), glucose (0.45-0.54 g/L), Na (116-121 meq/L), K (3.42-3.81 meq/L), Cl (100-116 meq/L), Ca (7.98-8.61 mg/dL), P (8.31- 9.36 mg/dL), Mg (2.26-2.55 mg/dL), Fe (105-178 ug/dL), ALP (144-170 IU/L), ALT (10.0-14.8 IU/L), AST (42.8-53.4 IU/L), GGT (7.8-10.6 IU/L), LDH (99-135 IU/L), CHE (151-185 IU/L) and CPK (365-500 IU/L), were obtained. Some parameter ranges were similar to those obtained in amphibians, birds or mammals; others were very different. These parameters are useful to evaluate sanitary, metabolic and nutritional state on captive bullfrogs

Con el propósito de obtener valores normales sanguíneos y urinarios, 302 muestras de ejemplares sanos de Rana catesbeiana del nordeste argentino (9-21 meses de edad, 50-350 g de peso vivo, 50% de cada sexo), fueron analizados por espectrofotometría, electroforesis, densitometría, refractometría y microscopía. Fueron obtenidos intervalos de confianza (p<0.05) para hematocrito (28.6-31.6%), eritrocitos (0.40-0.44 T/L), VCM (686-732 fL), hemoglobina (6.41-7.20 g/dL), HCM (151-164 pg), CHCM (22.6-24.0%), leucocitos (18.7-22.3 G/L), neutrófilos (58.4-63.4%), linfocitos (23.9-29.8%), monocitos (2.1-3.8%), eosinófilos (4.6-7.0%), basófilos (2.9-4.1%), tiempo de sangría (289-393s), tiempo de coagulación (452- 696s), tiempo de protrombina (76-128s), densidad urinaria (1.0061-1.0089 g/mL), pH urinario (6.38-6.96), fibrinógeno (0.59-0.99 g/dL), proteínas totales (4.19-4.49 g/dL), albúmina (1.49-1.67 g/dL), alfa-1 globulina (0.20-0.24 g/dL), alfa-2 globulina (0.48-0.54 g/dL), beta globulina (0.68-0.77 g/dL), gamma globulina (1.28-1.42 g/dL), relación albúmina/globulinas (0.50-0.58), creatinina (4.09-5.56 mg/L), urea (76.1-92.4 mg/L), ácido úrico (11.5-15.4 mg/L), triglicéridos (0.34-0.52 g/L), colesterol total (0.56-0.67 g/L), C-HDL (0.03-0.05 g/L), C-LDL (0.34-0.44 g/L), alfa lipoproteína (6.01-8.67%), beta lipoproteína (91.3-93.9%), glucosa (0.45-0.54 g/L), Na (116-121 meq/L), K (3.42- 3.81 meq/L), Cl (100-116 meq/L), Ca (7.98-8.61 mg/dL), P (8.31-9.36 mg/dL), Mg (2.26-2.55 mg/dL), Fe (105-178 ug/dL), ALP (144-170 IU/L), ALT (10.0-14.8 IU/L), AST (42.8-53.4 IU/L), GGT (7.8-10.6 IU/L), LDH (99-135 IU/L), CHE (151-185 IU/L) y CPK (365-500 IU/L). Algunos intervalos fueron semejantes a los obtenidos en anfibios, aves o mamíferos, pero otros resultaron muy diferentes. Estos parámetros son útiles para evaluar estados sanitario, metabólico y nutricional de la rana toro en cautiverio

Plasma retinoid profile in bullfrogs, Rana catesbeiana, in relation to agricultural intensity of sub-watersheds in the Yamaska River drainage basin, Québec, Canada.

Amphibian populations are decreasing globally and the causes are presently unclear. Retinoids have been extensively studied in other vertebrate classes where they are associated with pleiotropic effects such as susceptibility to disease (including cancer and parasitic infections), deformities and reproduction. To investigate the hypothesis that retinoid homeostasis is influenced by agricultural activities, blood samples were collected from adult bullfrogs, Rana catesbeiana, at each of six sub-watersheds chosen to represent a gradient of agricultural intensity within the Yamaska River drainage basin. Samples of surface water were collected at each of the study sites approximately 1 month after spraying and analyzed for 53 pesticides. Male body weight was significantly different (p<0.001) between study sites with the smallest bullfrogs captured from the Rivière à la Barbue sub-watershed associated with high agricultural intensity. A significant linear regression (p<0.001; R2=0.176) was obtained between plasma retinol and body weight. Plasma retinol concentrations were significantly different between study sites (p<0.001) being lowest at both Rivière Noire and Rivière à la Barbue. More than 60% of the land area in these sub-watersheds is under intensive corn-soya cultivation and surface water contained the highest concentrations of the herbicides atrazine, deethyl-atrazine, simazine, metolachlor, dimethenamide, chlopyralide, dicamba and bentazone. Plasma 13-cis-4-oxo-retinoic acid was significantly different (p<0.001) between sub-watersheds, however this effect was apparently unrelated to agricultural intensity. Plasma retinol was negatively correlated (p=0.026; r=-0.237) with plasma 13-cis-4-oxo-retinoic acid. These results suggest that retinoid homeostasis in bullfrogs may be influenced by agricultural practices.

Blood and urine physiological values in farm-cultured Rana catesbeiana (Anura: Ranidae) in Argentina.

A total of 302 samples of healthy farm-cultured Rana catesbeiana specimens (9-21 months-old, 50-350 g liveweight, 50% each sex) from the north-east of Argentina, were analyzed through spectrophotometry, electrophoresis, densitometry, refractometry and microscopy in order to obtain blood and urine normal values. Confidence intervals (p<0.05) for PCV (28.6-31.6%), RBC (0.40-0.44 T/L), MCV (686-732 fL), hemoglobin (6.41-7.20 g/dL), MCH (151-164 pg), MCHC (22.6-24.0%), WBC (18.7-22.3 G/L), neutrophils (58.4-63.4%), lymphocytes (23.9-29.8%), monocytes (2.1-3.8%), eosinophils (4.6-7.0%), basophils (2.9-4.1%), bleeding time (289-393s), coagulation time (452-696s), prothrombin time (76-128s), urinary density (1.0061-1.0089 g/mL), urinary pH (6,38-6.96)., fibrinogen (0.59-0.99 g/dL), total protein (4.19-4.49 g/dL), albumin (1.49-1.67 g/dL), alpha-1 globulin (0.20-0.24 g/dL), alpha-2 globulin (0.48-0.54 g/dL), beta globulin (0.68-0.77 g/dL), gamma globulin (1.28-1.42 g/dL), albumin/globulin ratio (0.50-0.58), creatinine (4.09-5.56 mg/L). urea (76.1-92.4 mg/L), uric acid (11.5-15.4 mg/L), triglycerides (0.34-0.52 g/L), total cholesterol (0.56-0.67 g/L), HDL-C (0.03-0.05 g/L), LDL-C (0.34-0.44 g/L), alpha lipoprotein (6.01-8.67%). beta lipoprotein (91.3-93.9%), glucose (0.45-0.54 g/L), Na (116-121 meq/L), K (3.42-3.81 meq/L), Cl (100-116 meq/L), Ca (7.98-8.61 mg/dL). P (8.319.36 mg/dL), Mg (2.26-2.55 mg/dL), Fe (105-178 ug/dL), ALP (144-170 [U/L), ALT (10.0-14.8 IU/L), AST (42.8-53.4 IU/L), GGT (7.8-10.6 IU/L), LDH (99-135 IU/L), CHE (151-185 lU/L) and CPK (365-500 IU/L), were obtained. Some parameter ranges were similar to those obtained in amphibians, birds or mammals; others were very different. These parameters are useful to evaluate sanitary, metabolic and nutritional state on captive bullfrogs.

Influence of the photocycle and thermocycle on rhythms of plasma thyroxine and plasma and ocular melatonin in late metamorphic stages of the bullfrog tadpole, Rana catesbeiana.

The diel fluctuations in plasma thyroxine (T(4)) and plasma and ocular melatonin entrain to the light/dark (LD) cycle in the bullfrog tadpole, although the phase of the rhythms changes during development. Previous studies on the rhythmicity of these hormones were conducted under various LD cycles, but with a constant temperature, raising the question of the role of the natural thermocycle in determining the phase of the rhythms, and the changes that occur in the hormone levels and rhythms during late metamorphosis. To study this question, tadpoles were acclimated to simulated natural conditions of 14.5L:9.5D with a corresponding thermocycle in which the thermophase was 28 degrees C and the cryophase was 18 degrees C, or to the same thermocycle under constant light (24L). On both photoregimens, the diel fluctuations changed between prometamorphosis and metamorphic climax. However, more statistically significant rhythms, as indicated by the cosinor, occurred on 14.5L:9.5D than on 24L. At climax on the LD cycle, all hormones peaked around the same time in the late scotocryophase, whereas on 24L, plasma T(4) peaked in the thermophase and plasma and ocular melatonin peaks occurred some distance from each other early in the cryophase. The earlier peaks of plasma and ocular melatonin on 24L were due to a transient rise in these hormones at the onset of the cryophase, which was not sustained in the absence of an LD cycle. On 14.5L:9.5D with a corresponding thermocycle, the hormone rhythms had nearly the same phases as was found in previous work on 12L:12D at a constant temperature of 22 degrees C, allowing for minor phase shifting due to the photocycle differences, indicating that in this species laboratory studies on constant temperature give valid results even in the absence of a thermocycle. The findings show that the phases of the hormone rhythms are determined by the LD cycle although the onset of the cryophase, in the absence of a photocycle, may exert some influence on the nighttime rise in melatonin. The developmental rise in plasma T(4), and drop in plasma melatonin, occurred on both 14.5L:9.5D and 24L, indicating, taken together with previous work, that these climactic changes were independent of temperature and light cycling.

Developmental and diel profiles of plasma corticosteroids in the bullfrog, Rana catesbeiana.

Corticosteroids synergize with the thyroid hormone (TH) at late metamorphic stages and might have a role in the hormonal regulation of amphibian metamorphosis. This role could be influenced by diel fluctuations, particularly if the peak of the plasma corticoids changed in relation to the TH peaks. Diel variation in plasma corticosteroids was studied in Rana catesbeiana prometamorphic and climax tadpoles on 18:6, 12:12 and 6:18 light:dark (LD) cycles. Cortisol (hydrocortisone; HC) and aldosterone (ALDO) exhibited different, but LD cycle-specific, circadian fluctuations at prometamorphosis, whereas corticosterone (CORT) was undetectable (less than 1.18 ng/ml). HC, ALDO and CORT rhythms became synchronous at early metamorphic climax on all LD cycles, although the cosinor-derived acrophases, which occurred around the time of the dark:light transition, shifted approximately 6 h earlier from 18L:6D to 6L:18D. On both 18L:6D and 12L:12D, the acrophase of HC changed little from prometamorphosis to climax, whereas that of ALDO underwent a major phase shift. On 6L:18D, both the ALDO and the HC acrophases shifted at climax. These LD cycle-specific phase shifts of the diel rhythms placed the acrophases of the corticoids in different phase relationships to that of the previously determined thyroxine (T(4)) acrophase at climax, and may partially explain the influence of the light regimen on metamorphic timing. The pronounced diel variations in the corticoid concentrations from the troughs to the peaks show that hormone levels are a function of the time of day and the environmental lighting regimen, which need to be taken into account in measuring the level of plasma hormones in amphibians. The 24-h means calculated from the data of all the sampling times showed that only plasma ALDO and CORT, but not HC, rose markedly at climax, although there were significant LD cycle-related differences in the mean levels of both HC and ALDO at prometamorphosis, and in HC at climax. Additional work sampling at mid-light showed that plasma CORT peaked at Stage XXIII, decreased at the end of climax, and remained low in the postmetamorphic froglet at 2.1 ng/ml. In the adult bullfrog, CORT was clearly the predominant corticosteroid at 34.3 ng/ml, whereas HC and ALDO levels were only approximately 1.3 ng/ml.

Developmental and diel changes in plasma thyroxine and plasma and ocular melatonin in the larval and juvenile bullfrog, Rana catesbeiana.

Diel variation in plasma thyroxine (T(4)), and plasma and ocular melatonin was studied in Rana catesbeiana tadpoles and postmetamorphic froglets on 12:12 and 6:18 light/dark (LD) regimens. A progressive rise in plasma T(4) initiates metamorphosis while melatonin can modulate metamorphic progress. Changes in the phase of the rhythms of these two hormones during development might influence the hormonal regulation of metamorphosis. The hormones studied exhibited LD cycle-specific diel fluctuations except in froglet plasma T(4) and all hormones at prometamorphosis on 6L:18D. On 12L:12D, plasma T(4) and ocular melatonin peaked during the scotophase at prometamorphosis and early climax, whereas the plasma melatonin acrophase shifted from the light to the dark at climax. A nocturnal peak of plasma melatonin closely correlated with the onset and offset of dark appeared in the froglet, while the peak of ocular melatonin shifted to the light. Compared to 12L:12D, the peaks of the diel fluctuations on 6L:18D occurred later than on 12L:12D in synchrony with an earlier onset, and increase in length, of the scotophase. The phase of the hormone rhythms changed during metamorphosis in such a way that the peaks of melatonin had a different relationship to the T(4) peaks as development proceeded. On both LD cycles, the 24-h mean of plasma T(4) rose at climax and fell in the froglet whereas plasma melatonin decreased at climax and then rose to a high level in the froglet. After only minor changes during metamorphosis, froglet ocular melatonin levels decreased on 12L:12D and increased on 6L:18D. The findings indicate that the hormonal flux during metamorphosis has circadian aspects, which might explain variations in the response to exogenous hormone treatment at different times of the day and LD cycle-specific timing of development. A fall in plasma melatonin at climax appears to be as much a part of the hormonal changes of metamorphosis as a rise in plasma T(4).

Partial purification and characterization of 12-lipoxygenase in bullfrog erythrocytes.

12-Lipoxygenase (12-LO) in bullfrog (Rana catesbeiana) erythrocytes was purified partially by ion exchange chromatography and affinity chromatography. Bullfrog 12-LO was a single chain protein with a pI of 7.1-7.8 and MW of 7.77 kDa. This enzyme did not show typical Michaelis Menten type kinetics. At low substrate concentrations, it had a lag phase and at higher substrate concentrations, the activity was inhibited. The product of linoleic acid (LA), 13-hydroperoxy-9, 11-octadecadienoic acid (13-HpODE), was an activator for the enzyme. When arachidonic acid (AA) was used as substrate, 13-HpODE also affected the Km of bullfrog 12-LO towards AA. The affinity of LA towards bullfrog 12-LO was higher than the affinity of AA. Suicide inactivation was much more rapid than that of any mammalian 12-LO reported. Hemoglobin (Hb) inhibited the activity of 12-LO partially and removing Hb eliminated this inhibition. Both Hb and Met-Hb inhibited the 12-LO activity but did not denatured completely the Hb, suggesting that the inhibition was a direct interaction between 12-LO and Hb protein chain and was not due to competition between 12-LO and Hb for oxygen. This study characterizes bullfrog 12-LO with respect to stability, optimal pH, suicide inactivation and interaction with Hb and provides important evolutionary information about this enzyme.

Rapid and selective removal of larval erythrocytes from systemic circulation during metamorphosis of the bullfrog, Rana catesbeiana.

Mechanisms of hemoglobin transition during bullfrog metamorphosis were investigated by labeling red blood cells from larvae (L-RBC) and from froglets (A-RBC) with a fluorescent dye, PKH26. The life span of the labeled L-RBC in systemic circulation was significantly shorter when they were injected into the animals at the metamorphic climax, compared to injection into pre- or postmetamorphic animals. The A-RBC had a long life span regardless of the metamorphic stage of the recipient animal. Therefore, L-RBC were selectively removed from the systemic circulation at the time of metamorphic climax. During climax, the labeled L-RBC were ingested by hepatic and splenic macrophages, indicating that macrophages are involved in the specific elimination of L-RBC.

Serum chemistry and hematology values for anesthetized American bullfrogs (Rana catesbeiana).

Samples taken from seven male and seven female adult American bullfrogs (Rana catesbeiana) were evaluated by complete blood count and serum chemistry to establish baseline data on commercially available frogs destined for laboratory use. Differences between sexes were analyzed and females had higher plasma protein, calcium, and sodium levels.

Eicosanoid synthesis by purified thrombocytes and erythrocytes from warm- and cold-acclimated American bullfrogs (Rana catesbeiana).

Amphibian blood plays an important role in eicosanoid synthesis. Although clotting frog blood produces eicosanoids, the cellular source of prostaglandins and thromboxanes in bullfrog blood is unknown. Thromboxane (TX)B2 synthesis from purified thrombocytes was affected by 30-day cold-acclimation at 5 degrees, but not PGE2 or leukotriene (LT) synthesis. Although no cyclooxygenase activity has been found in human erythrocytes, frog erythrocytes were capable of forming cyclooxygenase products, but the amounts were lower than those produced by thrombocytes. Additionally, there was no effect of cold exposure on eicosanoid production by isolated erythrocytes. Similar to some mammalian nucleated white blood cells, nucleated bullfrog thrombocytes and erythrocytes produced leukotrienes. The production of eicosanoids by thrombocytes was stimulated by A23187 and thrombin. Erythrocytes were stimulated by A23187. Control synthesis by erythrocytes and thrombocytes was inhibited by 5 microM indomethacin (cyclooxygenase pathway) or nordihydroguaiaretic acid (5-lipoxygenase pathway) and cyclooxygenase products were increased in the presence of nordihydroguaiaretic acid. Thrombin stimulation of eicosanoid production by thrombocytes was inhibited when the inhibitors were present prior to the final centrifugation of the cell isolation. The results suggest that cold exposure can affect eicosanoid synthesis in thrombocytes, but not erythrocytes, and that thrombocytes are a major source of eicosanoids in bullfrogs. The production of cyclooxygenase and lipoxygenase products by nucleated erythrocytes and thrombocytes suggests a role for these compounds in hemostasis and inflammatory responses in these animals.

Thrombocytes are the predominant source of endogenous sulfidopeptide leukotrienes in the American bullfrog (Rana catesbeiana).

Nucleated bullfrog erythrocytes have 5-lipoxygenase (LO) and are the first non-mammalian cell to exhibit endogenous sulfidopeptide leukotriene (LT) synthesis. Non-nucleated mammalian platelets lack 5-LO, but contribute significantly to LTC4 production by transcellular synthesis. However, nucleated bullfrog thrombocytes have not been examined for 5-LO activity. Endogenous leukotriene synthesis by bullfrog thrombocytes and mixed leukocytes was analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC). Calcium ionophore activated (A23187) leukocytes demonstrated 5-LO, 12-LO, and 15-LO activity. Spectral analysis demonstrated synthesis of LTB4, LTB4 isomers, 15(S)-monohydroxyicosatetraenoic acid (HETE), 5(S),12(S)-diHETE, 5(S),15(S)-di-HETE, lipoxin A4 (LXA4) and LXB4. Thrombocytes synthesized large quantities of sulfidopeptide leukotrienes but no lipoxins. Sulfidopeptide leukotriene and LTB4 radioimmunoassay analysis and the radiological RP-HPLC profile of [3H]AA metabolism further confirmed synthesis. Incubations with [3H]LTC4 demonstrated slow and incomplete conversion to [3H]LTD4. Thrombocyte leukotriene profile changed over time revealing a significant shift from the LTC4 synthase to LTA4 hydrolase pathway, corresponding with release of large amounts of LTA4. Thrombocytes potentially play a pivotal role in inflammatory and cardiovascular responses. 5-LO activity in amphibian homologs to mammalian platelets and erythrocytes compared with the lack of activity in the mammalian counterparts may correspond to the loss of the nucleus in the evolution of these cells.

Effect of glucocorticoids on thyroid hormone action in cultured red blood cells from Rana catesbeiana tadpoles.

Metamorphosis in anuran amphibia requires thyroid hormone (TH) and can be induced prematurely by the administration of TH. There is also evidence that the developmental effects of TH in these forms are modified by other hormones. For example, PRL has been shown to retard and corticosterone (B) to accelerate some, but not all, components of TH-induced metamorphosis. Red blood cells (RBCs) of Rana catesbeiana tadpoles exhibit a 4- to 5-fold increase in thyroid hormone receptor (TR) number (sites per nucleus) in vivo during either spontaneous or TH-induced metamorphosis. In the present study this TH-induced effect on RBC TR number was examined in an in vitro culture system. RBC TR number was increased by T3 in vitro; the maximum effect (2-fold increase) was obtained after exposure to 0.3 nM T3 for 60 h. This T3-induced increase in TR number was completely abolished in the presence of either 34 nM B or 10 nM dexamethasone, whereas basal TR number was unaffected. The effect appears to be a specific effect of glucocorticoid (GC), because it was not mimicked by the sex steroid, testosterone, and it was not obtained when RU-486, a glucocorticoid antagonist, was included with B in the medium. Other experiments demonstrated that the T3-induced increase in RBC TR was associated with an increase in the TR alpha messenger RNA level. This increase in TR alpha messenger RNA was reduced, but not eliminated, in the presence of concentrations of GC that abolished the TH-induced increase in TR, suggesting that the effects of GC occur in part at a pretranslational level. Using a GC binding assay, tadpole RBCs were found to contain approximately 10(4) GC receptors/cell. These findings indicate that B may be a physiological modulator of TH action in tadpole RBCs. This inhibitory effect of GC contrasts with previous reports that GC accelerates some of the morphological effects of TH in developing tadpoles, indicating that the nature of this modulating effect on TH action is tissue specific.