Histopathological alterations in bullfrog juveniles fed commercial rations of different crude protein levels1

The relationship between the quality of dietary crude protein and health of bullfrog juveniles (Lithobates catesbeianus) was evaluated by necropsy and histopathological examination of animals. The histopathology results showed that animals fed different feeds, regardless of CP levels, presented lymphocytary hepatitis, colitis and flattened microvillosities; kidneys with areas of tubulonephrosis and renal tubule calcification; myocarditis and cardiac muscular fiber dissociation. Such injuries suggested a degenerative nutritional process, with development of inflammatory processes spread to all the organs. These findings suggested that the animals had been fed with proteins of low biological value, indicating, apparently, poor feed quality, that harmed the health of the frogs and, consequently, their performance. Complementary studies are necessary to understand the biochemical behavior of the bullfrog in its second phase of life, supplying data for a better understanding of the nutrition of these animals.


Introduction
Native to the cold regions in south Canada and the northern United States, the Bull-frog (Lithobates catesbeianus) has a peculiar characteristic: it grows and reproduces quickly.This characteristic makes it well adapted when captive.In its original habitat, it can reach 170 g in four years and its ideal weight for slaughter is from 170 to 200 g.However, when captive, it can reach this weight at 7 months old in the case of tropical regions, depending on the temperature, handling, feeding, and genetic potential (Lima & Agostinho, 1988).
Brazil is today the world's second largest producer of frogs in captivity, with proprietary technology for breeding and processing.There are aproximately 600 frog producers in various states of the country and a third of them are in the state of Rio de Janeiro (Mello et al., 2006).Some stages of management are involved in situations that interfere with the general health of animals, including nutrition, sanitary and livestock management, which, if neglected, lead to the death of several animals, causing great economic losses.Nutritional handling is the main cause of the problems in frogculture.Furthermore, the composition of an adequate nutritional feed is directly related to protein content and the quality of these proteins (Hipolito et al., 2004).
Nutritional and sanitary handling aim to boost breeding by using feed to obtain nutrients in desired quantities.However, the imbalance of the carbohydrate and protein ratio in the feed given to animals, or their possible substitutes, undermines the whole nutritional process and can lead to morphological cell changes in various organs of the animals (Hipolito et al., 2004).These morphological changes are important causes of adaptation, injury and death of the cell.There are five types of cellular adaptation: atrophy, hypertrophy, dysplasia, metaplasia and hyperplasia.The types of cellular injury are cell swelling, fatty metamorphosis, necrosis and apoptosis (Kumar et al., 2008).
Therefore, the objective of this study was to evaluate the systemic histopathological changes in bullfrog juveniles (Lithobates catesbeianus) fed commercial feed used by frog farms in Rio de Janeiro with different formulations and indications of three crude protein levels.

Material and Methods
The experiment was conducted by the AQÜISUAM Research Group in the Biology Research Laboratory of UNISUAM, Rio de Janeiro.During the experimental period of 60 days, 360 bull-frog juveniles (Lithobates catesbeianus Shaw, 1802) from spawning were used, with average weight of 14.955 g (± 1.3 g), distributed in twelve 1 m 2 tanks, with a density of 30 animais/m 2 under water in a semi-immersed pond system.
The water was distributed in the tanks by polyvinyl carbon pipes, with replacement of 100% volume every 24 hours during the experimental period, and flow through PVC small pipeline installed in the back of the tanks and connected to the sewer.The water level inside the tanks was maintained by a knee type device adapted in the back of the tanks.
Commercial diets were used (Table 1) with different formulations, with indications of three levels of crude protein, CP (28, 36 and 45%), each one with four replications, in a randomized complete design according to Seixas Filho et al. (1998).Forty eight bull-frog juveniles were used, three repetitions of each replicate per treatment, collected at the end of the experiment and subjected to autopsy, to verify histopathological changes in the liver, kidneys, intestines and heart.These organs were fixed in a metacarmim solution for 12 hours and kept in 70% alcohol.Subsequently, they were embedded in paraffin, cut 5 µm thick in microtome and stained with hematoxylin and eosin to observe the slides using light microscopy.

Results and Discussion
Primary or secondary nutritional disorders can lead to morphological changes which cause symptoms relatively late, although the appearance of morphological changes is not related to functional deficits, as they may arise well before anatomical changes are evident.
Macroscopically, the livers, were pale (data not shown), and in the histopathological examination, there was intense focal mononuclear inflamatory infiltrate in the subcapsular regions and hepatic lobules, in addition to venous congestion (Figures 1, 2 and 3).
These changes may be associated with the commercial formulations of the diets used, since they are designed for fish because there is no unique formulation for the bullfrog at any of its stages of development.The lack of a specific product may interfere with the nutritional process of bull-frog juveniles, because during this stage, the frogs have a digestive system suitable for carnivores, with short intestines.Because these diets tend to meet more of the characteristics o f omnivorous animals, the bull-frog juvenile does not adapt to this type of feed, which leads to a process of prolonged protein-calorie malnutrition (Hipólito, 2003).This process involves changes in liver function due to lack of good quality protein, which may cause serious injuries in this organ with clear reduction in the growth rate and death of the animal (Hipólito et al., 2001).Moreover, it is reported in the literature that the Mycotoxicoses, in association with the deterioration of the diet, may cause blood congestion, and focal areas of interstitial hepatitis and hepatitis (Hipólito et al., 2001).
Lesions were observed in the hindgut of animals characterized by mononuclear enteritis and colitis, with loss or flattening of the longitudinal folds and hyperplasia of epithelial cells (Figures 4, 5 and 6), when the intestinal structure of these animals was compared to healthy frogs (George et al., 1998).The loss or flattening of the longitudinal folds contradicts literature reports of a sharp reduction in the height of the intestinal villi in weaned piglets, caused by supplying a diet of low nutritional value (Lopes et al., 2004).Furthermore, it has been shown that adult bull-frogs which have suffered nutritional intoxication with protein deficiency present peeling and atrophy of the longitudinal folds of the intestinal epithelium (Hipólito et al., 2001).The intake of feed with low protein quality can cause lesions in the intestinal tissue caused by stress and infectious agents, in Figure 1 -Photomicrograph of liver image of bull-frog juveniles fed diets of 45% CP stained with HE, 100 X.
Presence of lymphocytic hepatitis with large numbers of mononuclear cells (arrow).The same lesions were observed in imagoes fed diets containing 28 and 36% CP.
Presence of subcapsular hepatitis (arrow) and venous congestion (A).The same lesions were observed in bull-frog juveniles fed with diets containing 28 and 36% CP.  addition to the development of an intense inflammatory response to fight these agents, which could, at least in part, explain the cases of enteritis and mononucleaer colitis.Microscopic examination of renal tissue showed tubule nephrosis, with tubular degeneration and destruction of tubular epithelial cells, which suggested renal failure (Figure 7), that may be associated with prolonged feeding Figure 4 -Photomicrography of hindgut of bull-frog juveniles fed diets of 45% CP stained with HE, 100 X.
Presence of colitis in lymphocyte proliferation (arrow) and proliferation of epithelial cells (A) and area with normal monolayer of epithelial cells (B).The same lesions were observed in bull-frog juveniles fed diets containing 28 and 36% CP.
Presence of hypertrophy and epithelial hyperplasia (arrow).Normal area (A).The same lesions were observed in juvenile bull-frog fed diets containing 28 and 36% CP.  with low quality protein, as it has been shown in humans that patients with short bowel syndrome from malnutrition develop a framework for renal failure, caused by gromerulonefrose (Durks et al., 2007).
In some cases, the bull-frog juvenile also showed tubular calcification (Figure 7), which can be explained by renal failure, which leads to renal decrease of Presence of tubule-nephrosis with tubular degeneration (A) and calcification of renal tubules (arrow).The same lesions were observed in bull-frog juveniles fed diets containing 28 and 36% CP. potassium, the main cause of renal hypercalcemia, and that change in the calcium metabolism may account for by the calcification observed in the kidneys of bull-frogs fed diets of low nutritional value (Rubin, 2006).Finally, the histopathology of heart tissue showed dissociation of myocardial fibers with focal areas of myocarditis, suggesting death of cardiomyocytes and congestive heart failure (Figures 8 and 9).One explanation for these changes may be the fact that the reduction in the absorption of proteins with high nutritional value leads to a reduction in the supply of iron to animals and can cause anemia, which would reduce the availability of oxygen in the blood and cardiac ischemia, a major cause of cardiomyopathy in mammals (Rubin, 2006).
Decoupling of the fibers (A) and normal area (B) of the myocardium.The same lesions were observed in bull-frog juveniles fed diets containing 28 and 36% CP.

Conclusion
The histopathological findings suggested that the animals were fed in a manner which is inappropriate to their digestive system and/or the feed contained proteins of low biological value, therefore with poor quality diets, which harmed their health and performance.

Figure 3 -
Figure 3 -Photomicrograph of liver image of bull-frog juveniles fed diets of 45% CP stained with HE, 200 X.

Figure 2 -
Figure 2 -Photomicrograph of liver image of bull-frog juveniles fed diets of 45% CP stained with HE, 100 X.

Figure 5 -
Figure 5 -Photomicrography of hindgut of bull-frog juveniles fed diets of 45% CP stained with HE, 200 X.

Figure 6 -
Figure 6 -Photomicrography of hindgut of bull-frog juveniles fed diets of 45% CP stained with HE, 200 X.

Figure 7 -
Figure 7 -Photomicrograph of kidney of bull-frog juveniles fed diets of 45% CP stained with HE, 100 X.

Figure 8 -
Figure 8 -Photomicrography of heart of bull-frog juveniles fed diet with 45% CP stained with HE, 100 X.

Figure 9 -
Figure 9 -Photomicrography of the heart of bull-frog juveniles fed diets with 45% CP stained with HE, 200 X.