Blood and Urine Physiological Values in Farm-cultured Rana Catesbeiana (anura: Ranidae) in Argentina

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The bullfrog, Rana catesbeiana Shaw 1802 (bullfrog) has its origin in North America.Specimens present in Argentina come from genetic lines imported from Brazil, and they are adapted to the tropical climate (Roman 1994).There are more than 200 bullfrog hatcheries in Argentina which produce meat marketed at a high price (Carnevia 1995).The edible meat is valued because of its low fat and cholesterol levels (Pavan 1996).The bullfrog is characterized by its size; in captivity it can reach 300 g liveweight after 12 months.Since aging causes a decrease in the food conversion index, frogs are sacrificed when they are 6-7 months old Indoor captivity is the system chosen to rear this animal as escapes would be dangerous to the ecosystem.R. catesbeiana is ecologically considered as an "undesirable guest".When it settles in any lagoon, the original aquatic fauna could rapidly become extinct due to the high food consumption (Lima and Agostinho 1992) of this species in which cannibalism would not be unusual (Longo 1985).In contrast, a natural diet for an autochthonous terrestrial anuran like Bufo sp. would be mainly composed of coleopterans and hymenopterans (Duré and Kehr 1999).
Blood and urine composition would be influenced by peculiar physiological characteristics of the amphibian, such as metamorphosis, water and solutes skin exchange, capacity to support hemodilution and hemoconcentration, modification of urinary bladder water permeability, metabolic and enzymatic changes due to temperature, fast during winter lethargy, and others (Goldstein 1982, Eckert 1992, Candia et al. 1997, Bicego and Branco 1999, Busk et al. 2000, Curtis and Barnes 2001).It has recently been discovered that adrenergic receptors redistribution (Bachman et al. 1998), as well as eicosanoids (Herman and Luczy 1999) and natriuretc peptides synthesis increase (Uchiyama et al. 1998) have active participation in acclimatization mechanisms and corporal fluid retention in R. catesbeiana.
Contrary to their close relatives (reptilians and birds) which are uricotelics, adult R. catesbeiana is ureotelic, although in tadpole stage it reveals an ammoniotelic pattern of nitrogen excretion (Goldstein 1982, Curtis andBarnes 2001).Frog blood is hyperosmotic in relation to the fresh water they live in, and urine is hyposmotic in relation to their blood (Coppo 2001).Corporal fluids pH varies according to body temperature, it is acidified when temperature increases and vice versa; exchange of Na between cell and internal environment is also altered by pH (Jorgensen 1995).Changes of pH provoke numerous hematic and urinary modifications (Coppo 2001).
Except in the case of plasmatic electrolytes, texts of animal physiology reveal a manifest absence of hematic and urinary normal values from amphibians diagnostic parameters.Such parameters would be useful to evaluate health state in captive R. catesbeiana, which can suffer malnutrition, anemia, stress, transmissible diseases, intoxications, hemorrhagic dysfunctions, as well as inflammation and necrosis of liver, lungs, kidneys, spleen, muscles and other organs (Fraser 1986, Lima and Agostinho 1992, Hecnar 1995, Goldberg et al. 1998, Fontenot et al. 2000).
The objective of this study was to obtain physiological reference values from hematic and urinary diagnostic parameters in R. catesbeiana.

Experimental subjects, feeding and handling:
A total of 302 healthy R. catesbeiana specimens were used for a period of two years; 270 of them were maintained on intensive culture systems, divided in 3 frog farms in the north-east of Argentina.Samples from 90 frogs (9-21 months old, 50-350 g liveweight, 50% each sex), were taken in each breeding place.Thirty six per cent of the samples was taken during winter time, and 64% during the remaining seasons.No heating system during winter season was used in the hatcheries; all of them supplied food (45% protein balanced pellets, milled bovine lung, worms and fly larvae) at a rate of 3-5% liveweight/day.The 32 remaining animals were reared on an extensive system (semi-captivity), in a closed lagoon where frogs selected exclusively "natural" food.They were adult 16-20 month-old animals from both sexes.Samples were taken during winter and all along the rest of seasons.
Sample Taking: Frogs were transported to the laboratory in thermal boxes which contained a NaCl 0.6% isotonic solution cooled with ice (2-3ºC); this procedure provokes desensitization and lethargy, facilitating the animal manipulation (Lima and Agostinho 1992).Liveweight was obtained in an electronic balance Scientech-SL, with a 0.01 g accuracy.Samples were taken in the morning (7-8 AM), after a 24 h fasting period.Blood was obtained by intracardiac puncture, carried out with syringe and needle.The sample was a venose and arterial blood mixture, since frogs, with their anatomical characteristic, possess a unique ventricle (Goldstein 1982).Some of the blood was treated with anticoagulant (EDTA, 0.34 mol/L), another was mixed with a sodium citrate solution (130 mmol/L) and the last one was centrifuged (700g, 10 min) to obtain serum.Urine was obtained by cystocentesis.
Laboratory procedures: Being amphibians erythrocytes nucleated cells, erythrogram parameters were obtained applying avian techniques (Coles 1989).There was a previous blood hemolysis and centrifugation to eliminate erythrocyte nuclei, and hemoglobin was later evaluated by photocolorimetry (Drabkin technique, using Wiener Lab reagents).Red blood cells (RBC) concentration was determined by means of Neubauer hemocytometer microscopic count using Biopur diluters, and the packed cell volume (PCV, hematocrit) was measured by capillary centrifugation (12 000 g, 5 min).White blood cells (WBC) concentration was obtained from stained smear count (Giemsa), considering corrections according to PCV value.Differential leukocyte count was carried out from stained smear (May Gründwald).Blood cells size was measured with an ocular micrometer.Erythrocyte indices such as mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and MCH concentration (MCHC), were obtained by conventional calculation.
Bleeding, coagulation and prothrombin time were evaluated respectively by Dukes, Lee-White and Quick methods (Pesce and Kaplan 1990).Fibrinogen was calculated by the difference between plasma and serum proteins (Coles 1989), using an Erma-D refractometer.Urinalysis (density, pH, sediment, and chemical composition) was carried out by conventional laboratory techniques (Coppo 2001).Sodium and potassium were evaluated using Biopur reagents, in a Metrolab 305-D flame photometer.
The separation of proteins (albumin and alpha, beta and gamma globulins, on cellulose acetate) and lipoproteins (alpha and beta, on agarose gel) was carried out by electrophoresis (Pesce and Kaplan 1990).Fractions were quantified in a Citocon CT-440 densitometer.

Statistical analysis:
The normality of the distribution of dependent variables (quantitative continuous) was assessed using the Wilk-Shapiro test (WS).Parametric descriptive statistics included measures of central tendency (arithmetic mean, x), dispersion (standard deviation, SD) and ranges.Fiduciary probability was assessed by confidence intervals (CI±95%).Correlation coefficients were obtained by the Pearson procedure.Calculations were all made using the Statistix software, 1996 version.

RESULTS
Values obtained from hemogram, coagulation tests, and some urinalysis parameters (Table 1), as well as from serum chemical values (Table 2), showed an approximately normal distribution, which allowed the use of parametric statistics.Confidence intervals were adjusted around arithmetic means, but individual ranges were wide.Correlation between age and weight was significant (r = 0.82, p = 0.02).
Chemical tests on urine revealed that 7.6% of the studied amphibians had protein vestiges (30 mg/dl) and 4% showed bilirubin traces, which coexisted with small quantities of ketones.In all cases glucose was negative.Hemoglobin vestiges were verified in 57.6% of the samples.Urobilinogen was found in 100% of the studied amphibian urinary samples, with concentrations of 0.2 mg/dl (92% of animals) and 1 mg/dl (8% of animals).Scarce quantities of erythrocytes (57.6% of cases), leukocytes (15.3%), germs (53.8%) and granular cylinders (8%), were verified in the urinary sediment.No crystals were found in these amphibians' urine.

DISCUSSION
After food ingestion, changes in amphibian plasma composition would be registered (Busk et al. 2000).Other changes would also occur as consequence of circadian rhythm, caused by cortisol fluctuations (Wright et al. 1999).Both postprandial and circadian effects were excluded from the present study design due to previous fast and basal condition of samples, and also because blood extraction was carried out in uniform morning hours.Scarce regulation mechanisms and higher tolerance to hemodilution and hemoconcentration, would cause a great oscillation of blood values in frogs (Goldstein 1982).This fact could explain the wide extent of ranges obtained in this trial.Correlation between age and weight was only moderately significant (r = 0.82, p = 0.02), probably because of the growth delay which takes place during the winter (Lima and Agostinho 1992).
Nutritional anemias would be common in amphibian.Coccidia, as Babesiosoma stableri, would be located inside erythrocytes; Lankesterella minima would also parasite tadpoles and adult frog RBC (Desser et al. 1990).Hematocrit would decrease in anemias, and it would increase in dehydration and postprandial stage.The latter would be due to spleen RBC release (Busk 2000).Hematocrit and hemoglobin would diminish as a consequence of alimentary deficiencies and prolonged fast (Singh 1978).Erythrocytes indicators in the nutritional state panel show a decrease owing to insufficient protein, vitamins B 12 , E, niacin and folic acid intake (Kolb 1987).Appropriate erythropoiesis would require a continuous and balanced affluence of minerals such as Fe, Cu, Co and Se; nutritional lacks would also provoke hematocrit and hemoglobin decrease (Jain 1993).

Leukogram:
The WBC concentration found on R. catesbeiana by Cathers 1997 (5.2±2.9G/l) was lower than the one obtained in the present trial (20.5±4.6 G/L), presumably because of a depression caused by the anesthetic used in sample taking.The WBC average found on these frogs coincides with the reference intervals reported on birds (13-22 G/L, Coppo 2001, and 18-30 G/L, Kolb 1987) but it is higher than those published on domestic mammals (6-12 G/L, Coppo 2001; 5-20 G/L, Kolb 1987, and7.6-16 G/L, Coles 1989).
Leukogram is useful to evaluate infection, inflammation, stress, neoplasias and other dysfunctions.Amphibian leukocytes may possess properties different to those of mammals, because temperature would greatly affect the cellular inflammatory response (Dias and Catao-Dias 1989).In R. catesbeiana tadpoles, lymphocytes and monocytes, but not granulocytes, would participate in the inflammatory focus development (Zablith et al. 1997).
The fundamental triad of hemostasis exploratory tests in human beings and domestic animals is constituted by bleeding, coagulation and prothrombin times, which evaluate platelet function, intrinsic pathway, and extrinsic pathway respectively (Coles 1989, Coppo 2001).In coagulative anomalies is also important to determine the plasma fibrinogen concentration, to discard eventual hypo-, dis-, and a-fibrinogenemias provoked by hemorrhagic diathesis, hepatopathies, and malnutrition (Kaneko 1989, Pesce andKaplan 1990).Amphibian thrombocytes would provide the necessary factors to form thromboplastin, which would transform fibrinogen into fibrin (Eckert 1992, Curtis andBarnes 2001).
The knowledge of coagulogram values from R. catesbeiana could contribute to clarify coagulopathies caused by inadequate diet, intestinal malabsorption, intoxications or metabolic disturbances (stress, cholestasis, fatty liver, myelodystrophies).Hypovitaminosis K provokes cutaneous hemorrhages as prothrombin time increases.Perhaps the most important bullfrog coagulopathy is the red-leg syndrome; it is a septicemia that causes skin and skeletal muscle hemorrhages, cutaneous ulcers, inflammation and necrosis of liver, spleen, and other celomic organs, emaciation, and death.Diverse intoxications and hepatopathies can lead to a deficit of fibrinogen and/or other clotting factors (Hecnar 1985, Fraser 1986, Lima and Agostinho 1992).
Water volumes equivalent to 30-50% of body weight can be stored in terrestrial amphibian urinary bladder, which is capable of absorbing water and salt against gradient, and cause urine hyposmolarity.Recent studies demonstrate that urinary vesical wall has the ability to regulate its water permeability (Candia et al. 1997).Protection against water loss is mainly based on the oliguria: urine will concentrate until it is isosmotic in relation to plasma.No amphibian can produce urine which could be hyperosmotic in relation to blood (Wilson 1989).Urine concentration mechanisms based on solutes resorption (until they are hypertonic to plasma), are characteristic of mammals, not amphibian.
Urinary pH registered in the present study was almost neuter (6.68±0.71).It is acid (up to 5) on carnivores, and alkaline on herbivores (up to 8.4) (Kaneko 1989, Coles 1989).Urinary pH would be from 5-8 on birds, diminishing up to 4.7 in aquatic species when they are submerged (Coles 1989).Glucose tubular resorption would be total in this species, because its presence in urine was not verified in any of the cases; glucosuria is abnormal in all domestic animals (Coles 1989, Kolb 1989).Urobilinogen found in the urine of the studied frogs would be normal, because it is the hemoglobin metabolism terminal product; it is habitually present in urine of both carnivorous and herbivores species (Coppo 2001).
On birds' urine, but not in those of mammals, it would be normal to discover vestiges of blood, bilirubin, protein and ketones (Coles 1989), as it found in these frogs.Germs and cylinders presence is abnormal on mammals' urine, but the existence of epithelial (genital and urinary) cells is usual, as well as some leukocytes, such as those found in frogs.The presence of abundant phosphate, carbonate and urate crystals reported respectively in carnivores, herbivores and birds (Coles 1989), contrasts with the absence of crystals in the urine of these frogs.Physicochemical characteristics verified on R. catesbeiana urine (low density, pH nearly neutral, absence of crystals) are in correspondence with those from species whose habitat facilitates the residues excretion without necessity of preserving great water quantities (Coppo 2001).
On mammals, all albumins and half of globulins that circulate in blood, are synthesized in the liver; on amphibians, this function would be carried out by the hepatopancreas (Goldstein 1982).Plasma proteins intervene in acid-base balance, immunity, coagulation, colloid-osmotic pressure, and blood viscosity; they also transport hormones, vitamins, lipids, bilirubin, calcium, zinc, iron and copper (Kolb 1987, Kaneko 1989).Albumins are excellent indicators of protein biosynthesis; they also operate as nutritional reserve of amino acids, which would be habitually exchanged between plasma and tissues, mainly in skeletal muscles (Coppo 2001).Proteinogram is of clinical interest because it facilitates the diagnosis towards alterations such as alimentary lacks, malabsorption, hepatopathies, inflammations, and renal, coagulative, and immunologic dysfunctions (Coles 1989, Pesce and Kaplan 1990, Coppo 2001).
These results confirm that R. catesbeiana is an ureotelic rather than uricotelic amphibian.Contrary to their uricotelic relatives (reptiles and birds), mature amphibian, as well as mammals, would excrete NPN in the form of urea, although in tadpole stage they would excrete ammonia (Eckert 1992).Exceptionally, some frogs (Phyllomedusa sauvagii and Chiromantis xerampelina) would excrete NPN in urate form, and some toads (Xenopus laevis) would be ureotelic during their permanency on earth, but they would become ammoniotelic when they are in water (Goldstein 1982).
In spite of their ureotelic pattern, amphibians would retain urea to regulate their osmotic pressure.Environment salinity increase would cause urea retention because it increases the urea hepatic synthesis and decreases the urea renal excretion.This fact could be proved in Rana cancrivora specimens exposed to fresh water versus sea water.They registered differences in plasma osmolarity (290 versus 830 mOsm/l), urea (40 versus 350 mMol/l), sodium (125 versus 250 mEq/l) and urine flow (100 versus 1%) respectively (Goldstein 1982).This clearly indicates that frogs utilize urea to maintain their hyperosmolarity with the environment (Wilson 1989).
Lipoprotein metabolism would reveal similar characteristics among different animal species, but it would not be exactly the same in all of them.Canine, feline, equine, ruminant and some rodents would have "HDL pattern", characterized by plasma alpha lipoprotein predominance.When these animals are fed on fatty diets, cholesterol is linked by HDL rather than LDL, avoiding noxious effects due to protective action attributable to HDL.Human beings, pigs, rabbits, marmots, and several monkey species, would respond to the "LDL pattern", because when they consume fat, they increase their beta lipoprotein and they are exposed to a major atherogenic risk (Bauer 1997, Coppo 2001).Bearing in mind that C-LDL level was higher than C-HDL level, and that alpha lipoprotein ratio was lower than beta lipoprotein ratio, frogs would join in the "LDL pattern" rather than the "HDL pattern".Similarly, other researchers found rates of LDL higher than HDL in R. catesbeiana plasma, although they also found vestiges of low density lipoprotein (VLDL), which was not detected in the present study (Suzuki et al. 1976).
Lipidogram values may vary due to age, heredity, food type, and diverse illnesses, such as hepatic and renal failure, malabsorption, stress, hypothyroidism, and infections.Cholesterol would rise in the initial phase of starvation (due to high fat mobilization), but in case of prolonged fast its plasma concentration tends to decrease (Kaneko 1989, Coles 1989, Pesce and Kaplan 1990, Coppo 2001).
In amphibians, glucemia would decrease during the stage previous to winter lethargy, with an increase of hepatic glycogen; hypoglucemia would cause hypothermia.Insulin would decrease glucemia and temperature in R. catesbeiana ; on the contrary, high temperature would increase the O 2 consumption and it would cause hyperglucemia (Rocha and Branco 1998).Plasma glucose would be regulated through insulin, glucagon, adrenaline, cortisol, and thyroid hormones (Curtis and Barnes 2001).Physiologically, glucemia might vary by effects of age and physical exercise; pathologically it would alter in malnutrition, stress, and endocrine and hepatic failures (Coles 1989, Kaneko 1989, Coppo 2001).
In frogs, electrolytes and water enter the organism through skin and digestive tract, being eliminated by skin, urine and feces; amphibians skin could check the osmolarity of the surrounding liquid (Wilson 1989).Fresh water frogs are hyperosmotic in its environment, that is the reason why they tend to incorporate water by the skin and decrease their corporal saline concentration (Goldstein 1982).In these animals, high internal osmolarity (210-290 mOsm/L) and low external osmolarity (50 mOsm/L), could provoke overhydration (the entry of water by osmotic gradient) and loss of electrolytes (diffusion by concentration gradient).Homeostasis is achieved with abundant hypotonic urine and an increase in electrolytes tubular resorption and salt cutaneous absorption (Eckert 1992).
Chronic hepatic disorders result in increased plasma ALP in most animals.During normal bone growth in young animals, a large amount of ALP is in plasma; osteopathies also results in increased plasma ALP.Recently, GGT has been found to be liver specific and is used as an indicator of hepatobiliary disease.Increased plasma AST is associated with cell necrosis of the liver and skeletal or cardiac muscle, starvation and lack of vitamin E. ALT is well established as a marker of acute hepatic damage.Injury to skeletal and cardiac muscle results in considerable increase in plasma CPK.Brain also contains great amounts of the latter.LDH is released after cellular damage to the liver, lung, muscle, heart and kidney tissue.CHE is originated in liver, pancreas, intestinal mucosa and brain; decrease in CHE has been reported in liver failure, muscular dystrophy, chronic renal disease and organophosphate insecticide intoxication (Coles 1989, Kaneko 1989, Pesce and Kaplan 1990, Coppo 2001).
The obtained data could be useful to optimize the diagnosis of sanitary, metabolic and nutritional dysfunctions in R. catesbeiana .It could also cooperate with the search of real nutritional requirement of this amphibian in captivity.Such knowledge may lead to an improvement in frog meat production, thus a promising future.Frog meat world consumption ranges between 30 000 and 50 000 tn/ year, with an existing market for skin (leather crafts), intestine (esthetic surgery thread), liver (foie gras elaboration), and fat for cosmetic use (Lima and Agostinho 1992, Roman 1994, Pavan 1996).
Some hematic and urinary physiological values from R. catesbeiana were similar to those reported on other frogs (PCV, Na, K, Ca, Mg), and toads (total protein, albumin, alpha-1, alpha-2 and beta globulins, uric acid, triglycerides, glucose, P, Fe).In spite of the close phylogenic relationship between amphibians and birds, some parameters were quite different (RBC, hemoglobin, MCV, lymphocytes, creatinine, glucose, ALP, urinary density and sediment).Several frog blood values were similar to those found in human beings (ALT, GGT, bleeding and coagulation time), and both domestic monogastric (neutrophils, lymphocytes, LDL-C, Cl, LDH) and polygastric mammals (fibrinogen, AST).