Abstract

Pale liver syndrome (PLS) is a descriptive term for a collection of findings that occur with nutritional disease conditions of hatchery-reared Florida largemouth bass.^1-3 Pale liver syndrome is believed to reduce fingerling stocking survival rates.^1,3 Previous studies have eliminated toxins, bacterial, viral, and parasitic pathogens as primary causes of this condition.^1-3 Nutritional causes are associated with diets either having available carbohydrate content greater than 25%² and/or a fat content of greater than 12%, depending on the fatty acid profile.¹

The carbohydrate-associated condition is believed to be a glycogen storage disease (Bass-GSD) similar to mammalian metabolic disorders such as human-GSD type VI (insufficient liver phosphorylase activity). In bass, however, the cause is attributed to high intakes of available carbohydrate leading to progressive accumulation of glycogen in the hepatocytes.² This accumulation can be large enough to cause massive liver necrosis and nodular regeneration.² Liver nodules that result from Bass-GSD are reported not to progress to hepatocellular carcinoma (HCC).²

Liver lesions induced by bass diets containing high levels of fat with nutritionally poor fatty acid profiles are considered to be bass fatty liver disease (Bass-FLD).¹ High-fat diets (> 15% fat) containing concentrations of linoleic acid > 7% and stearic acid > 12% are found to be associated with multiple focal nodular hyperplasia (MFNH) and HCC.¹ Other lesions found in Bass-FLD include steatosis, lobular inflammation, swelling of hepatocytes, fibrosis, and focal inflammatory lesions. These lesions are progressive in development and severity.¹ Linoleic acid has a high correlation with the development of the most severe lesions, both MFNH and HCC. Stearic acid appears to be an agonist in the development of these lesions while oleic acid appears to be an antagonist.

The present study was designed to determine the effects of adding energy in the form of various dietary fats to high protein (60%), low carbohydrate (11%) diets. Diet composition was correlated with the development of liver lesions (Table 1) and utilization of nutrients on the growth and development of lesions (Table 2). Six experimental groups containing 250 fingerling bass each were used. Physical characteristics of the bass were: total length 12 +/- 2 cm, body condition (K) 1.17 +/- 0.05, and growth 0.82 +/- 3 mm/day. Analysis of variance showed that physical characteristics of the groups were not significantly different (p<0.05). Six experimental diets were prepared, one for each group. Dietary carbohydrate concentrations of the diets were 10.9 +/- 0.7%, dietary protein concentrations were 60 +/- 2% and dietary fat concentrations were 17.6 +/- 2.9%. Dietary fatty acid concentrations ranged between diets as follows: palmitic-myristic, 18 to 24%; arachidic-behenic-lignoceric, 0.6 to 1.0%; palmitoleic, 5.2 to 9.5%; stearic, 2.4 to 16.4%; oleic, 23 to 35% and linoleic, 3.6 to 4.4%. Caloric content per 100 g diet ranged from 401 to 425 calories.

The groups were maintained on the experimental diets for 63 days. Five whole fish from each of the six groups were collected at the end of the trial for proximate and fatty acid analysis. Five liver samples were collected from each group for histopathology, protein, fat, and fatty acid analysis. Liver physiological response variables included: liver size, hepatosomatic index (HSI), liver fat, prevalence of eosinophilic granule cells (EGC), focal inflammatory lesions (FIL), inflammation (INF), nodules and HCC. Explanatory variables used were growth rate, condition (K), feed gain, calories/100g (cal/100g) and concentrations of the following dietary nutrients: protein, fat, carbohydrate, ash, total saturated fatty acids, palmitic acid (C16), stearic acid (C18), palmitoleic acid (C16:1), total monounsaturated fatty acids, total saturated fatty acids >C16 [stearic (C18), arachidic acid (C20), behenic acid (C22) and lignoceric acid (C24)], oleic acid (C18:1), linoleic acid (18:2), total omega-3 and total polyunsaturated fatty acids.

Table 1. Positive Correlation¹ of Dietary Fatty Acid Concentrations with Liver Lesions

^1. Estimated by Pearson Correlation Coefficients, r>0.29

Death rates were less than 1% for the experimental period and 8 months following termination of the trial. Based on this study, a diet with a fat content of 20% with < 5% linoleic acid, fatty acid profiles similar to those of the experimental diets, and available carbohydrate concentrations of < 12% can be used to produce 100 mm bass fingerlings without severe PLS. All experimental fish had a mild steatosis, some inflammation and FIL, but neither nodules nor HCC were detected, probably because of the short 63 day time for development.

Increased dietary fat concentrations above 15% may cause a decrease in feed required for gain but it does not produce a significant increase in growth rate. Increasing dietary fat above 15% can be done without severe PLS (bass-GSD and bass-FLD) provided carbohydrate concentrations are below 10% and linoleic acid concentrations are below 5%. Dietary fat concentrations above 20% will probably require reduced concentrations of saturated fatty acids greater than 16 carbons to avoid bass-FLD. We are not certain of the availability of the carbohydrates in this trial but carbohydrate concentrations strongly correlated with growth rate (0.75) and the efficiency of carbohydrate utilization was >89%.

Table 2. Negative Correlation¹ of Nutrient Utilization Parameters² with Liver Lesions

^1. Estimated by Pearson Correlation Coefficients, r <-0.30
^2. (Nutrient Recovered from whole fish/nutrient fed) X100

The data indicated that palmitic, palmitoleic, oleic, omega-3, and total polyunsaturated fatty acids did not significantly correlate with an increase in the incidence of liver lesions with the exception of slight increases in liver size. Concentrations of saturated fatty acid greater than 16 carbons in length did significantly correlate with inflammation (INF) and focal inflammatory lesions (FIL, Table 1). Stearic acid was the major contributor to the significant correlation of saturated fatty acid concentrations with INF and FIL. Based on these results stearic acid (C18), arachidic acid (C20), behenic acid (C22) and lignoceric acid (C24) are all considered agonists in the incidence of severe liver lesions for high fat diets (Table 2). Efficiency of utilization data indicated that only stearic acid of saturated fatty acids greater than 16 carbons is synthesized in the bass. Therefore, reduced dietary concentrations of saturated fatty acids >C16 and replacement with oleic and palmitoleic acid would seem to be the best strategy to increase dietary fat above 20%.

The negative correlation of linoleic acid and saturated fatty acids with reduced metabolite utilization by the liver suggests a reason for the PLS reduction in survival rate of stocked bass fingerlings.

References

1.  Cardeilhac, P., H. Dickson, R. Stout, and S. Hardin. 2004. A pelleted feed for the high-volume production of largemouth fingerlings free of pale liver syndrome. Abstr Proc Int Assoc Aquat Anim Med; Pp. 12-13.

2.  Goodwin, A. E., R.T. Lochmann, D.M. Tieman, and A.J. Mitchell. 2002. Massive hepatic necrosis and nodular regeneration in largemouth bass fed diets high in available carbohydrates. J World Aquaculture Soc 33: 466-477.

3.  Porak, W., W. Johnson, S. Crawford, D. Renfro, T. Schoeb, R. Stout, R. Krause, and B. DeMauro. 2002. Factors affecting survival of largemouth bass raised on artificial diets and stocked into Florida lakes. Am Fish Soc Symp 31: 649-665.