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Brazilian Journal of Animal and Environmental Research 
ISSN: 2595-573X 
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Brazilian Journal of Animal and Environmental Research, Curitiba, v.7, n.2, p. 1-14, 2024 
Biochemical analysis of bullfrog liver and blood: insights into growth and 
development 
 
Análise bioquímica do fígado e do sangue de rã-touro: insights sobre 
crescimento e desenvolvimento 
 
Análisis bioquímico del hígado y la sangre de la rana toro: el crecimiento y el 
desarrollo 
 
DOI: 10.34188/bjaerv7n2-033 
 
Submetido: 19/01/2024 
Aprovado: 01/03/2024 
 
Adriana Sacioto Marcantonio 
Doutora em Aquicultura pelo Centro de Aquicultura da UNESP / CAUNESP Jaboticabal 
Agência Paulista de Tecnologia dos Agronegócios - APTA Regional - Unidade de Pindamonhangaba 
Pindamonhangaba, SP. Brasil 
E-mail: adriana.marcantonio@sp.gov.br 
 
Matheus Phelipe Marin 
Graduando em Agronomia pela universidade de Taubaté – Unitau, SP. 
Universidade de Taubaté 
Pindamonhangaba, SP. Brasil 
E-mail: matheusophellipe@hotmail.com 
 
Erna Elisabeth Bach 
Doutora em Agronomia-Fitopatologia/ USP/ESALQ/Depto Fitopatologia. 
Instituto Biológico – APTA - SAA 
São Paulo, SP. Brasil 
E-mail: ernabach@gmail.com 
 
Fernanda Menezes França 
Doutora em Ciências pela Unidade de São Paulo - USP/Escola de Engenharia de Lorena 
Instituto de Pesca – APTA – SAA 
São Paulo, SP. Brasil 
E-mail: fernanda_ranicultura@yahoo.br 
 
Mikel Eduardo de Mello 
Mestre em Aquicultura e Pesca - Instituto de Pesca. 
Instituto de Pesca – APTA – SAA 
São Paulo, SP. Brasil 
E-mail: mikeleduardo@yahoo.com.br 
 
Rafael Lopes Faria 
Mestrando do Instituto de Pesca 
Instituto de Pesca - APTA - SAA 
São Paulo, SP. Brasil 
E-mail: rafalopesfariaa@gmail.com 
 
Claudia Maris Ferreira 
Doutora em Ciências pela Faculdade de Medicina da Universidade de São Paulo – FMUSP 
Instituto de Pesca – APTA – SAA 
São Paulo, SP. Brasil 
E-mail: cmferreira@sp.gov.br 
Brazilian Journal of Animal and Environmental Research 
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Brazilian Journal of Animal and Environmental Research, Curitiba, v.7, n.2, p. 1-14, 2024 
ABSTRACT 
The aim of this study was to evaluate the productive and physiological performance of bullfrogs 
(Lithobates catesbeianus) after metamorphosis, through zootechnical growth parameters and the 
quantification of proteins and enzymes present in the liver. In addition we observed the behavior of 
circulating amino acids in the blood. The experiment was conducted at the Experimental Station of 
the Aquaculture, APTA Regional – SAA, in São Paulo, Brazil. We used 400 L. catesbeianus froglets 
weighing 5.76±1.32g, which were distributed in four fattening tanks in the wet system, installed 
under an agricultural greenhouse at density of 51 frogs/m². The animals were fed with 5% of the 
biomass of each tank every day, with extruded feed for carnivorous fish with 40% Crude Protein. 
Every 28 days, they were weighed to calculate weight gain and apparent feed conversion ratio. At 
the same time, two animals from each tank were chosen randomly to have their blood and liver 
collected for biochemical analysis to quantify amino acids, proteins, phenols, peroxidase and 
polyphenol oxidase. The results obtained indicate that the absence of screening every 15 days 
affected the development and uniformity of the animals, favoring cannibalism. The average 
apparent feed conversion was 1.84. There was an increasing and proportional formation of proteins 
with low degradation as the animals gained weight. As for amino acids, we detected arginine, 
isoleucine, alanine and tryptophan in the first month, with trypsin appearing after 60 days. These 
results lead us to state that total or partial screening is essential during the first month after 
metamorphosis, and that the feed provided, although not specific to the species, fulfilled its role in 
animal metabolism, with little formation of phenolic compounds and free radicals. 
 
Keywords: frogculture, feed conversion, weight gain, ranaculture, Lithobates catesbeianus 
 
RESUMO 
O objetivo deste estudo foi avaliar o desempenho produtivo e fisiológico de rãs-touro (Lithobates 
catesbeianus) após a metamorfose, por meio de parâmetros zootécnicos de crescimento e da 
quantificação de proteínas e enzimas presentes no fígado. Além disso, observamos o 
comportamento dos aminoácidos circulantes no sangue. O experimento foi realizado na Estação 
Experimental de Aquicultura, APTA Regional - SAA, em São Paulo, Brasil. Foram utilizadas 400 
rãs L. catesbeianus pesando 5,76±1,32g, que foram distribuídas em quatro tanques de engorda no 
sistema úmido, instalados sob uma estufa agrícola com densidade de 51 rãs/m². Os animais foram 
alimentados diariamente com 5% da biomassa de cada tanque, com ração extrusada para peixes 
carnívoros com 40% de proteína bruta. A cada 28 dias, eles foram pesados para calcular o ganho de 
peso e a taxa de conversão alimentar aparente. Ao mesmo tempo, dois animais de cada tanque foram 
escolhidos aleatoriamente para terem seu sangue e fígado coletados para análise bioquímica para 
quantificar aminoácidos, proteínas, fenóis, peroxidase e polifenol oxidase. Os resultados obtidos 
indicam que a ausência de triagem a cada 15 dias afetou o desenvolvimento e a uniformidade dos 
animais, favorecendo o canibalismo. A conversão alimentar aparente média foi de 1,84. Houve uma 
formação crescente e proporcional de proteínas com baixa degradação à medida que os animais 
ganharam peso. Quanto aos aminoácidos, detectamos arginina, isoleucina, alanina e triptofano no 
primeiro mês, com o aparecimento de tripsina após 60 dias. Esses resultados nos levam a afirmar 
que a triagem total ou parcial é essencial durante o primeiro mês após a metamorfose, e que a ração 
fornecida, embora não específica para a espécie, cumpriu seu papel no metabolismo animal, com 
pouca formação de compostos fenólicos e radicais livres. 
 
Palavras-chave: cultivo de rãs, conversão alimentar, ganho de peso, ranicultura, Lithobates 
catesbeianus 
 
RESUMEN 
El objetivo de este estudio fue evaluar el desempeño productivo y fisiológico de ranas 
toro(Lithobates catesbeianus) después de la metamorfosis, mediante parámetros zootécnicos de 
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crecimiento y la cuantificación de proteínas y enzimas presentes en el hígado. Además se observó 
el comportamiento de los aminoácidos circulantes en la sangre. El experimento fue realizado en la 
Estación Experimental de la Acuicultura, APTA Regional - SAA, en São Paulo, Brasil. Utilizamos 
400 ranitas L. catesbeianus con peso de 5,76±1,32g, que fueron distribuidas en cuatro tanques de 
engorde en el sistema húmedo, instalados bajo un invernadero agrícola a densidad de 51 ranas/m². 
Los animales fueron alimentados diariamente con el 5% de la biomasa de cada tanque, con pienso 
extrusionado para peces carnívoros con un 40% de Proteína Bruta. Cada 28 días, se pesaron para 
calcular la ganancia de peso y el índice de conversión alimenticia aparente. Al mismo tiempo, se 
eligieron al azar dos animales de cada tanque a los que se les extrajo sangre e hígado para realizar 
análisis bioquímicos con el fin de cuantificar aminoácidos, proteínas, fenoles, peroxidasa y 
polifenoloxidasa. Los resultados obtenidos indican que la ausencia de cribado cada 15 días afectó 
al desarrollo y uniformidad de los animales, favoreciendo el canibalismo. La conversión alimenticia 
aparente media fue de 1,84. Hubo una formación creciente y proporcional de proteínas con baja 
degradación a medida que los animales ganaban peso. En cuanto a los aminoácidos, detectamos 
arginina, isoleucina, alanina y triptófano en el primer mes, apareciendo tripsina a los 60 días. Estos 
resultados nos llevan a afirmar que el cribado total o parcial es esencial durante el primermes tras 
la metamorfosis, y que el pienso suministrado, aunque no específico para la especie, cumplió su 
función en el metabolismo animal, con escasa formación de compuestos fenólicos y radicales libres. 
 
Palabras clave: ranicultura, conversión alimenticia, ganancia de peso, ranacultura, Lithobates 
catesbeianus 
 
 
1 INTRODUCTION 
The first specimens of the bullfrog, Lithobates catesbeianus (Frost et al., 2006; AmphiaWeb, 
2022), arrived in Brazil in 1935 from Canada. Frogculture began in Brazil in this year with the 
import of 300 bullfrog couples by the Aurora Frog farm, located in the state of Rio de Janeiro 
(Ferreira et al., 2002). Unlike other countries that practice extensive frog cultivation or legalized 
hunting, Brazil has developed captive rearing technologies, which have relied on the efforts of 
farmers, research institutions and universities (Cribb et al., 2013). Even though the animal originated 
in North America, it has adapted to Brazil's climate, showing hardiness, good adaptation to different 
physical and dietary management, as well as reaching sexual maturity in less than a year, and is the 
only species used by Brazilian commercial farms (Ferreira et al., 2002; DIAS et al., 2009). With the 
decline in natural frog stocks due to predatory hunting, the legal ban on hunting and growing 
demand, frogculture has gained popularity, positioning the country as the largest producer in the 
Americas (Cribb et al., 2013). 
One of the main challenges of raising frogs in commercial farms is to eliminate the stress 
factors (predators, competition for food and space) that, both in the wild and in captivity, slow down 
growth, which in turn could direct all its energy towards fattening and/or reproduction, through 
appropriate management and facilities and the use of balanced and nutritious food (REIS et al., 
2022). 
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The bullfrog's diet is based on the quantity and quality of the protein in the feed. The frog 
begins life as a tadpole, with a tail, no legs and a short, oval body. Gradually, it loses its tail and 
develops into a set of four legs. Once these changes are complete, the animal is called a froglet, i.e. 
a fully formed frog with a body similar to that of an adult, but sexually immature. In Brazil, L. 
catesbeianus reaches sexual maturity at one year old or less and can have two or more spawnings 
per year (Cribb et al., 2013). On average, it takes three to four months for the animal to reach 
slaughter weight, making for a seven-month cycle. This variation depends on the temperature, 
management, feeding, genetic potential and rearing system (Castro et al., 2014; Pahor-Filho et al., 
2015; Mello et al., 2016). For decades, the amphibian system has been the most widespread 
production system used by Brazilian farmers. However, like any other branch of animal production, 
it needs adjustments, standardization and a strong reflection on the results obtained so far. The big 
challenge is to minimize the production costs of a carnivorous animal, making its rearing more 
economically viable (Pahor-Filho et al., 2019). In search of this economic viability, various feed 
formulations have been tested. However, excessive levels of protein or amino acids can affect the 
animal's metabolism, with the liver being the most affected organ. For perfect animal husbandry 
conditions, good liver function is essential, and the biochemical study of these functions helps 
diagnose disease and mortality situations in frog farming (Hipolito et al., 2001). 
For these reasons, and to contribute to the advancement of frogculture, the aim of this work 
was to evaluate the productive and physiological performance of bullfrogs (L. catesbeianus) after 
metamorphosis, by quantifying the proteins and enzymes present in the liver, as well as observing 
the behavior of circulating amino acids in the blood. 
 
2 MATERIALS AND METHODS 
The experiment was carried out during the summer in the Southern Hemisphere, in the 
Experimental Station of the Aquaculture Sector of the Regional Research and Development Unit – 
URPD, in Pindamonhangaba, Brazil, respecting animal welfare practices, and was authorized by 
the Animal Ethics Committee of Fisheries Institute. 
We used bullfrog froglets in the early stages of development (average weight = 5.76 ± 1.32g), 
which were distributed in four tanks (i.e. replicates) of the fattening system known as the "Wet 
System" (Cribb et al., 2013), measuring (1.50m x 1.30m = 1.95m²), installed under an agricultural 
greenhouse , covered with shade and plastic. The stocking density used was 51 animals/m², with 
100 animals per stall and 400 animals in total. The experimental period was 84 days. The animals 
remained submerged in water up to their heads (on average 0.05m, depending on the size of the 
frog) and captured the food which was thrown in and remained floating on the water in the tank. It 
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should be noted that the wet system does not use fly larvae for feeding, as the feed is supplied 
directly into the water (Zangerônimo et al., 2002). 
The water used to supply the tanks came from a dam. The animals were fed daily, but the 
feed supply was adjusted every 28 days according to their live weight (5% of biomass). Extruded 
feed for carnivorous fish was used with the following guarantee levels: 40% Crude Protein (Laguna 
- Socil), with 6mm, 6% Ethereal Extract, 5.5% Crude Fiber, 12% Mineral Matter, as proposed by 
LIMA et al. (2003). The tanks were cleaned daily and any dead animals removed. 
To assess production performance, we determined the zootechnical parameters of survival, 
cannibalism, weight gain and apparent feed conversion. To calculate weight gain, we used the 
formula WG = FW - IW, where WG is Weight Gain, FW is Final Weight and IW is Initial Weight. 
Feed Conversion Ratios (F.C.R) was determined using the formula: F.C.R = AFS / WG, where AFS 
is the amount of feed supplied (g) and WG is the weight gain (g). 
The animals were screened every 28 days, that is, separated by size to reduce the occurrence 
of cannibalism. The frogs were removed from the tanks, separated by size (small, medium and 
large), counted, weighed and redistributed in the tanks, respecting the proximity of the initial density 
used. Initially (zero moment 1) and before the screening, two animals were collected from each tank 
(n=8) for biochemical analyses showing the profile of amino acids, proteins, phenols and liver 
peroxidase. 
The amino acid profile was determined in peripheral blood samples collected from the 
animals using heparinized syringes. Before extraction, a local anesthetic (lidocaine™) was applied 
to the animal's hind leg so that it would not feel pain (Figure 1). The samples were centrifuged at 
2000 x g for 5 minutes to separate the plasma, and kept in the freezer until the biochemical tests 
were carried out. 
 
Figure 1 - Collection of peripheral blood from bullfrog froglet (Lithobates catesbeianus) using heparinized syringes for 
amino acid quantification. 
 
 
1 Zero Moment - Phase in which the froglets were in the begining of experimente. Fifiteen days old on average, after 
metamorphosis. 
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For amino acid analysis, the methodology described by Badawy & Morgan (1991) was used, 
where 0.4mL of pure Milliq water was added to 500uL of plasma, which was precipitated with 
0.1mL of 60% HCLO4. After filtering, 20uL was injected into the high-pressure liquidchromatography (HPLC) equipment. The HPLC is a YL-9300 coupled to a UV detector, wave 
length 254nm and with a temperature maintenance tower (27-30 °C) for a LUNA C18 column, 
reverse phase size 25cm x 4.5mm. All the plasma samples and standards were evaluated by the peak 
output in the HPLC separation and correlated with the retention time (Rt) confirmed by the Clarity 
software. 
After blood extraction, the animals were euthanized under deep anesthesia in a standard 
solution of eugenol, as it is popularly known, to remove the liver. Eugenol was previously diluted 
in alcohol (8mL eugenol diluted in 50mL alcohol) and then 4mL of this solution was used for each 
liter of water to be applied to the animals (Viriato et al., 2021). In addition, to complete the sacrifice 
of the animals, the spinal column was cut. Subsequently, the liver organ was removed using scissors 
and tweezers. The liver was then weighed, photographed and frozen to quantify proteins, phenols 
and peroxidase (Freitas et al., 2018 and Mascaro et al., 2014). They were then ground in a mortar 
and pestle with phosphate buffer pH=7 0.1mol/L, at a concentration of g/mL. 
The Shapiro-Wilks and Bartlett tests were applied to check the normality and 
homoscedasticity of the data. The analysis of variance (ANOVA) followed by the Tukey test was 
used to assess differences in performance averages (weight gain and feed conversion). Differences 
were considered significant when PIt is capable of showing signs of organic disorder such as nutritional deficiency, poisoning, 
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infection or parasitism, through changes in its cellular, biochemical and morphological structure 
(Freitas et al., 2018). This organ reflects the health and general condition of the animal, that is its 
main function is to metabolize, store and filter blood. The secretion of bile and functions related to 
most metabolic systems such as carbohydrates, fats and proteins, in addition to the miscellaneous 
storage of vitamins, blood clotting factors and iron storage. When there is a process arising from 
intoxication, the lesions have a pattern between them and this may indicate a condition represented 
by food, with the body needing to isolate toxic products that appear through cell disruption (Reis et 
al., 2022). 
It is in the liver that oxidative stress occurs, and. the formation of reactive oxygen species 
(ROS) and this happens whenever there is a physiological imbalance, due to the type of diet or 
exposure to adverse chemical substances. This imbalance results in the production of free radicals 
and damage to cellular components such as proteins, lipids and nucleic acids (Dornelles & Oliveira, 
2014). The body can survive without liver functions for carbohydrates and fat, but it would not 
survive without liver functions for proteins, such as deamination of amino acids, formation of urea 
to remove ammonia, formation of plasma proteins, interconversion between different amino acids 
and synthesis of non-essential amino acids. Amino acids are molecular structures with roles in 
protein formation, hormone synthesis, immune responses, antioxidants and other physiological 
functions (Hoseini et al., 2020). They are necessary for growth and are classified as non-essential, 
that is, those that can be produced by organisms, and essential, those that cannot be synthesized by 
animals. Essential amino acids must be systematically supplied from the diet. For aquatic organisms, 
arginine, alanine, leucine, isoleucine and trypsin play a fundamental role in growth, excretion 
metabolism and energy homeostasis (Calheiros et al., 2019; Li et al., 2009). 
In anurans reared in captivity, weight gain can be influenced by climatic conditions and the 
frogs' metabolism can vary according to the ambient temperature as they are ectothermic animals 
(Lima et al., 2003). In this study, the fact that the tanks were located inside an agricultural 
greenhouse covered with plastic and shade provided a constant temperature, with no sudden drops, 
which favored the performance shown in the animals' weight gain. However, the F.C.R was strongly 
influenced by cannibalism, which is often not a good result for the farmer. However, it remained 
within economically viable conditions for the farmer, as described by MOREIRA et al. (2013), who 
indicate that frog farming is attractive when feed conversion is around 2:1. 
To check physiological performance, we analyzed the amount of proteins, phenols, 
peroxidase and polyphenol oxidase. These molecules are interconnected and reflect the activity of 
the liver in relation to the food consumed and/or the elements of the environment detoxified by the 
animal, since frogs capture water by osmolarity. Our data shows a gradual increase in proteins over 
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the experimental period, with a peak in phenols and peroxidase at 60 days. Phenols are acidic 
compounds that, in large quantities, affect the liver, kidneys and nervous system, and peroxidase is 
an enzyme that catalyzes oxidation reactions and its increase indicates that there is an increase in 
free radicals (Hipolito et al. 2007). Fortunately, this situation stabilized after 90 days, indicating the 
balance and homeostasis of the animals' liver functions. 
When assessing this performance using blood plasma, four types of amino acids were 
detected at zero moment, when the animals were on average 15 days after metamorphosis. Two of 
them were essential (isoleucine and tryptophan) and two non-essential (arginine and alanine), the 
latter of which must be present in the feed. At 60 days after metamorphosis, the amino acid tyrosine 
was detected, which in turn was not detected at 90 days. Tyrosine, a constituent of most proteins, 
whose function is to help build muscle fibers and the structure of various organs, as well as other 
occasional essential amino acids, can disappear in situations caused by certain pathologies. On the 
other hand, at 90 days we began to detect valine (non-essential), which could be explained by the 
feed provided. 
We can conclude that total or partial screening is essential during the first month after 
metamorphosis, and that there is increasing and proportional protein formation as the animals gain 
weight. The feed provided, although not specific to the species, fulfilled its role in animal 
metabolism, with little formation of phenolic compounds and free radicals. 
In this study we have provided valuable information on the physiological and productive 
performance of bullfrog tadpoles after metamorphosis, as well as insights into the biochemical 
composition of the liver and blood. However, we suggest for future studies research related to 
species-specific diets comparing their impact on the animals' performance, as well as expanding 
analyses with biomarkers and biochemical compounds to provide a more comprehensive 
understanding of the animals' physiological state. 
 
ACKNOWLEDGEMENTS 
 
We would like to thank CNPq for awarding us the Scientific Initiation grant and the staff of the 
Experimental Station of the Aquaculture Sector of the Pindamonhangaba Regional Research and 
Development Unit for their hospitality. 
 
 
 
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