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.

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.

Endogenous sulfidopeptide leukotriene synthesis and 12-lipoxygenase activity in bullfrog (Rana catesbeiana) erythrocytes.

Endogenous leukotriene (LT) synthesis by mammalian inflammatory cells requires both 5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein. Other myeloid cells, like erythrocytes, have an incomplete 5-lipoxygenase pathway and synthesize leukotrienes transcellularly. Several studies indicate that sulfidopeptide leukotrienes have important physiological functions in bullfrogs and receptors have been characterized. Calcium ionophore activated bullfrog blood was analyzed by reverse phase-high-performance liquid chromatography (RP-HPLC). Endogenous metabolites consisted of 5-LO products including leukotriene D4. Other metabolites also suggested 12-lipoxygenase activity. Following purification, metabolites from activated erythrocytes were analyzed by RP-HPLC coupled with radioimmunoassay. Erythrocytes demonstrated endogenous synthesis of LTD4 which was inhibited by non-selective (NDGA) and specific (MK886) 5-lipoxygenase inhibitors. Experiments with partially purified erythrocyte cytosol further confirmed 5-LO activity and revealed 12-lipoxygenase activity. HPLC analysis of [1-14C]arachidonic acid labeled metabolites from activated erythrocytes indicates that most of the available substrate is converted to 12-hydroxy-eicosatetraenoic acid (12-HETE). These novel findings indicate that, in contrast to mammals, bullfrog erythrocytes endogenously synthesize LTD4 and large quantities of 12-HETE giving them the potential to contribute directly to inflammatory responses. The evolutionary loss of the nucleus in mammalian erythrocytes appears to be associated with the inability to synthesize leukotrienes endogenously.

Hemoglobin transition in early development stages of the tadpole, Rana catesbeiana.

Five hemoglobin components (a, I, II, III and IV) were isolated from the hemolysates of the tadpole, Rana catesbeiana. Component a was monomeric molecule and the other four were tetrameric molecules. Component I predominating in younger tadpoles was replaced by component II during tadpole development. Electrophoretic and chemical analyses on the constituent globin chains revealed that component a was very similar to the alpha-chains of components I and II, ad that components I and II differed from one another in their beta chains.

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.

Regulation of c-erbA-alpha messenger RNA species in tadpole erythrocytes by thyroid hormone.

Putative thyroid hormone (TH) nuclear receptors have been detected in several tissues of Rana catesbeiana tadpoles. T3 receptor number (sites per nucleus) in red blood cells (RBCs) and tail increases substantially just before metamorphic climax or in response to exogenous TH; in contrast, receptor number in liver remains relatively constant. TH receptors in mammals and birds are thought to be encoded by a c-erbA gene. In the present study, two c-erbA cDNAs, one prepared from Xenopus laevis oocytes (XenTR alpha 1) and one prepared from Rana catesbeiana tail (RC12), were used to examine the c-erbA-related mRNA species in Rana catesbeiana tissues and determine their role in the TH induction of tadpole RBC receptor number. XenTR alpha 1 encodes a protein with T3-binding properties typical of TH receptors. RC12 is almost 99% homologous with XenTR alpha 1 at the amino acid level and contains all of the putative T3-binding region and most of the DNA-binding region. Using either cDNA as a probe, it was found that two major species of c-erbA-related mRNA species (2.6 and 4.0 kilobases) were clearly evident in tadpole RBCs, tail, and liver. A third, more diffuse band (approximately 5.0 kilobases) was observed in RBC and tail. In RBCs, but not in liver, the combined level of c-erbA-related mRNA species was increased during spontaneous metamorphosis or after administration of TH. Furthermore, the TH-induced increase in both c-erbA-related mRNA species and receptor number in RBCs was prevented if actinomycin-D was administered with TH.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Putative nuclear triiodothyronine receptors in tadpole erythrocytes during metamorphic climax.

The characteristics of the nuclear T3 receptors present in red blood cells (RBCs) of Rana catesbeiana tadpoles undergoing metamorphic climax have been investigated with a T3 saturation technique. Because there were significant amounts of receptor-bound endogenous hormone, prolonged incubation in vitro (48 h at 21 C) was necessary to achieve binding equilibrium. Receptor number, which averaged 783 +/- 35 (+/- SE) sites/nucleus during early prometamorphosis (stages XIV-XVI), increased rapidly during the subsequent stages of this phase. By stage XIX, receptor number had reached a maximum of 2464 +/- 152. During climax, receptor number decreased steadily, and by stage XXV, it was lower than that observed during premetamorphosis (stages V-X). This decrease was attributed to the replacement of larval RBCs, which have a high receptor content, by adult RBCs, which possess significantly fewer sites per nucleus. At midclimax, adult and larval RBCs were separated on a Renografin continuous density gradient. Adult cells constituted more than 65% of the total cell population and were shown to contain only 126 +/- 46 sites/nucleus. Although the striking increase in receptor number preceded the substantial increases in plasma T4 and T3 levels that occurred during climax, it was associated with a measurable increase in plasma thyroid hormone levels. Furthermore, receptor number in stage XIV tadpoles was increased markedly after immersion for 14 days in water containing sufficient T3 to raise plasma T3 only to levels normally observed in late prometamorphosis. These findings strongly support the hypothesis that the increases in receptor number and plasma thyroid hormone levels that occur before climax in this species are causally related.

Rana catesbeiana tadpole red blood cells express an alpha, but not a beta, c-erbA gene.

Thyroid hormone (TH) receptor number in red blood cells (RBCs) from Rana catesbeiana (RC) tadpoles increases 4-fold during both spontaneous and TH-induced metamorphosis, an effect that we have previously shown to be preceded by an increase in the level of c-erbA-related mRNA. The goals of the present study were to obtain an RC c-erbA alpha cDNA that contains the entire open reading frame for a putative TH receptor protein, to determine if this protein has characteristics typical of a TH receptor, and to assess its contribution to the developmentally related increase in TH receptor number. To accomplish this, the missing 5'-sequence of a previously isolated partial RC c-erbA alpha cDNA (RC12) was synthesized by polymerase chain reaction (PCR) and spliced to RC12 to yield a 1490-basepair cDNA (RC15) that contained the entire coding sequence of the receptor protein. Transcription of RC15 followed by translation of its mRNA in a rabbit reticulolysate system yielded a 50-kilodalton protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein binds T3 with high affinity (Kd, approximately 0.1 nM), and its affinity for T3 is at least 5 times that for T4. The results of cotransfection studies indicate that RC15 can function as a TH receptor; when COS cells were cotransfected with a construct consisting of RC15 cloned in the expression vector CMV4 and TK28 mult, a construct containing rat GH gene TH response element sequences up-stream of a chloramphenicol acetyltransferase reporter gene, chloramphenicol acetyltransferase activity is expressed in the presence, but not in the absence, of T3. To determine whether RBCs contain any c-erbA beta mRNA transcripts that might contribute to the developmentally related increase in the transcripts detected using RC c-erbA alpha cDNAs, alpha- and beta-specific cDNAs were synthesized by PCR and used as probes in a variety of hybridization assays. In all experiments using conditions in which c-erbA beta transcripts were detectable in other tissues, there was no evidence that tadpole RBCs contained such species. Lack of any beta-specific transcripts was confirmed by PCR, using as template cDNA prepared by reverse transcription of RC RBC RNA. It was also noted that the RBC at metamorphic climax is the tissue with the highest content of alpha-specific c-erbA transcripts. It is concluded that the c-erbA alpha gene encodes a TH receptor, and that only the alpha-gene is expressed in tadpole RBCs and subject to regulation during development and by TH.

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.

Chemical characterization of the serum very low density and high density lipoproteins from bullfrog, Rana catesbeiana.

The chemical properties of very low density and high density lipoproteins of adult bullfrog serum were determined. This serum contained extremely low levels of both very low density lipoprotein (10-30 mg/100 ml) and high density lipoprotein (5-10 mg/100 ml). The constituents of very low density lipoprotein, on a weight percentage basis, were found to be 48.1% triglyceride, 17.3% cholesterol ester, 8.8% cholesterol, 11.6% phospholipid, and 12% protein. These constituents were also present in high density lipoprotein with weight percentage values of 3.7%, 19.3%, 11.9%, 25.2%, and 36.8%, respectively. The fatty acid compositions of the triglycerides, cholesterol esters, and phosphatidylcholine were quite similar in the very low density lipoprotein and high density lipoprotein. However, shingomyelin fatty acid composition was appreciably different in the two lipoproteins. Disc gel electrophoresis in sodium dodecyl sulfate-polyacrylamide gels produced patterns with one major (approximate molecular weight, 7,000) and several minor bands for the apoprotein of very low density lipoprotein and one major (approximate molecular weight, 28,000) and several minor bands for that of high density lipoprotein.

Chicken gonadotropin-releasing hormone II increases plasma catecholamines in the bullfrog.

The aim of the present experiments was to test the effects of two native neuropeptides, [His5,Trp7,Tyr8]gonadotropin-releasing hormone (chicken GnRH II) and [Trp7,Leu8]GnRH (salmon GnRH), on the sympathoadrenal system of chronically cannulated, conscious bullfrogs (Rana catesbeiana). We observed that i.v. injection of chicken GnRH II or salmon GnRH increased plasma noradrenaline and adrenaline concentrations, at doses that did not significantly affect arterial blood pressure or heart rate. Chicken GnRH II was 10 times more potent than salmon GnRH for increasing plasma adrenaline, while the two neuropeptides were equally effective in raising noradrenaline concentration. These observations are consistent with a regulatory role for chicken GnRH II in the bullfrog sympathoadrenal system.

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.

Temperature coefficient of and oxygen effect on the antimony microelectrode.

With regard to pH measurement of biological fluids in vivo with metal-metal oxide microelectrodes, the effect of temperature and partial pressure of oxygen on antimony (Sb) microelectrodes was examined, and pH of blood was estimated in the bullfrog. The temperature coefficient (dE/dt) of electromotive force (EMF) of Sb-microelectrodes in the range of 7 to 37 degrees C was -1.18 +/- 0.113 mV/degrees C (mean +/- SEM) in Ringer solution, whereas that of the pH glass electrode in the same solution was -0.43 +/- 0.035 mV/degrees C. When estimated in Tris buffer solution, it was -0.06 +/- 0.063 mV/degrees C for Sb-microelectrodes and 1.05 + 0.036 mV/degrees C for glass electrodes. The change of slope constant (alpha in -mV/pH) in the Sb-microelectrode due to temperature change could be predicted empirically from: alpha = 0.40 (t-25) + 55.3, where t represents the measuring temperature in degrees C. The resultant deviation of pH reading between Sb and glass electrodes, delta pHSb-Glass, may be expressed by: delta pHSb-Glass = 0.00183 (t-25) +0.016. In the range of 45 to 760 mmHg of oxygen partial pressure it fixed pH, the EMF increased linearly with the increase of Po2, the slope (dE/dlog(Po2)) being 11.7 +/- 0.42 (SEM) mV (n = 13, t = 25 degrees C). In consideration of the above effects, the blood pH of bullfrog was estimated to be 7.697 +/- 0.092 (SD) and 7.729 +/- 0.111 with glass and Sb-microelectrodes respectively, the difference between the two being relatively minor.

Kallikrein generates angiotensin II but not bradykinin in the plasma of the urodele, Amphiuma tridactylum.

Incubation of heat-denatured plasma from the urodele, Amphiuma tridactylum (three-toed amphiuma) or from the anurans Rana ridibunda (European green frog) and Rana catesbeiana (American bullfrog) with either glass beads, porcine pancreatic kallikrein or trypsin did not generate bradykinin-like immunoreactivity. However, peptides were generated in kallikrein-treated amphiuma plasma that contracted vascular rings from the bullfrog systemic arch and had a spasmogenic action on the bullfrog urinary bladder. These peptides which were not generated in trypsin-treated plasma, were purified to homogeneity by reverse-phase HPLC and their primary structures established as: Asp-Arg-Val-Tyr-Val-His-Pro-Phe ([Asp1,Val5]angiotensin II) and Asn-Arg-Val-Tyr-Val-His-Pro-Phe ([Asn1,Val5]angiotensin II). Incubation of synthetic [Asn1,Val5]angiotensin II with amphiuma plasma resulted in deamidation to [Asp1,Val5]angiotensin II. The data suggest, therefore that amphiuma plasma contains an L-asparagine amidohydrolase (asparaginase), as previously described for the eel. Although bradykinin-related peptides have been isolated from frog skin, this study provides evidence tha the kallikrein-kinin system may be absent from the blood of amphibia.

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.

[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.