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Effect of storage temperature on the quality of diets for the prawn, Penaeus monodon Fabricius
Aquaculture, 80 (1989) 87-95 Elsevier Science Publishers B.V., Amsterdam
87 -
Printed
in The Netherlands
Effect of Storage Temperature on the Quality of Diets for the Prawn, Penaeus monodon Fabricius MARGARITA
C. DE LA CRUZ, GREGORIA
ERAZO and MYRNA N. BAUTISTA
Aquaculture Department, Southeast Asian Fisheries Development Center, Tigbauan, Zloilo (The Philippines) (Accepted
14 October 1988)
ABSTRACT De la Cruz, M.C., Erazo, G. and Bautista, M.N., 1989. Effect of storage temperature of diets for the prawn, Penaeus monodon Fabricius. Aquaculture, 80: 87-95.
on the quality
The effect of storage temperature was evaluated on the basis of growth response of prawns fed for 10 weeks with diets stored at 0°C lO”C, 28”-31°C (ambient temperatures) and 40°C for a period of 10 weeks. Prawns were stocked at 15 pieces per 60-l oval tank supplied with water at 28°C and 32 ppt in a flow-through aerated system. There were five replicate tanks per treatment. Lowest weight gain (20 g) was observed for prawns fed the diet stored at 40°C and significantly higher growth response was observed as the storage temperature decreased (30.2 g at 28-31°C; 37.7 g at 0 ’ C and 10 ’ C ) . Body size was significantly (P < 0.05) affected by diet after 6 weeks of feeding and highly significantly (PC 0.01) after 8 weeks of culture. Peroxide values for diets exposed for 10 weeks to 28” -31 “C (12.8 meq/kg) and 40°C (15.0 meq/kg) were significantly higher than those exposed to 0” and 10°C (2.9 meq/kg). The highest survival rate (76%) and feed conversion (8.9%) were observed for prawns fed diets stored at low temperatures (0” or 1O’C). Severe necrosis of the hepatopancreatic cells was observed in P. monodon fed with diet stored at the high temperature.
INTRODUCTION
Most fish farmers do not realize the importance of proper feed storage. Often, they are more concerned with seasonal fluctuations in availability and price of feed, and resort to bulk purchases which are stored for long periods. During prolonged and improper storage, adverse physical conditions (moisture, heat, light ) and microorganisms (molds, bacteria, yeast) may cause deterioration of feed quality. The resulting decrease in palatability and nutritive value, including deterioration of amino acids, vitamins and fat (Chow, 1980), can lead to economic losses. The rate of oxidation of fat is greatly influenced by storage temperature and a rise of 10’ C approximately doubles this rate (Kulikov, 1978). Oxidation of fat gives rise to a feedstuff of lower biological energy value (Rum-
0044-8486/89/$03.50
0 1989 Elsevier Science Publishers
B.V.
88
sey, 1980) that may cause a reduction in growth of the animal (Stuart et al., 1985 ) . The use of deteriorated feeds also can result in increased disease outbreaks including one known as “lipoid liver degeneration” (Roberts, 1978). Travis (1955) considered the hepatopancreas as a storage organ of organic and mineral reserves. It is also a monitoring organ for the nutritional value of diets for P. monodon (Vogt et al., 1985). Since several authors (Johnson, 1980; Pascual et al., 1983; Vogt et al., 1985; Baticados et al., 1987) have reported the importance of the hepatopancreas in evaluating the nutritional value of the diet, it is imperative to include examination of this organ in assessing the quality of diets. The study reported here was conducted to determine the degree of rancidity of diets, and the growth, survival, and histopathological effects on the hepatopancreas of P. monodon juveniles fed diet stored at varying temperatures. MATERIALS AND METHODS
A practical diet for prawns (Table 1) was prepared by mixing all the dry ingredients in a Hobart mixer for 10 min. The oil was gradually added to the mixture and mixed for another 5 min and then a sufficient quantity of warm water to give a smooth consistency was added to the oil mixture and blended for another 5 min. A Hobart meat grinder with a 2-mm die was used for diet extrusion. The extruded moist pellets were broken into small pieces, divided into four batches, and stored in plastic bags at four different temperatures: 0’) 10’) 28-31’) and 40’ C for a period of 10 weeks. The proximate analysis of the diet was performed using standard methods ( AOAC, 1975 ) . Three hundred and sixty P. monodon juveniles with a mean weight of 1.5 g and standard deviation of 0.5 g were obtained from SEAFDEC ponds at Leganes, Iloilo, The Philippines, and acclimatized for 1 week under laboratory conditions while being fed crustacean pellets. The prawns were randomly distributed to 20,60-l tanks provided with continuous aeration and seawater filtered through sand and gravel at a rate of 0.8-1.0 l/min. Temperature and salinity ranged from 28” to 29°C and 32 to 34 ppt, respectively. Prawns were stocked at 15 pieces per tank, with five replicate tanks per treatment. Five PVC pipes, about 6 in (15 cm) long and 2 in (5 cm) in diameter were placed in each tank to provide cover and prevent cannibalism. Feeding of the test diet was done twice a day at 10% of body weight for the first 2 weeks, after which the feed was reduced gradually to 2% at 10 weeks. The amount fed was adjusted after biweekly sampling of prawns when the prawns were weighed individually. Faeces and uneaten feed were siphoned out every morning. Mortalities and molt stages were noted daily. The oval tanks were thoroughly cleaned when the prawns were removed for weighing every 2 weeks. Specific growth rate was computed as SGR = log weight (final ) - log weight
89 TABLE 1 Composition of experimental diet Ingredients
g/100 g dry diet
Fish meal Squid meal Shrimp head meal Soybean meal (defatted) Rice bran Bread flour Cod liver oil Soybean lecithin Vitamin mix’ Mineral mix’ Cholesterol
15 15 15 15 14 15 5 2 1.5 1.5 1.0
Nutrients
Proximate composition (% as feed)
Moisture Crude protein Crude fat Crude fiber NFE Ash
7.15 45.02 17.6 4.20 24.51 9.11
IVitamin mix (mg/lOO g dry diet); p-aminobenzoic acid, 3.16; biotin, 0.10; inositol, 126.18; nicotinic acid, 12.62; calcium pantothenate, 18.93; pyridoxine HCl, 3.17; riboflavin, 2.57; thiamine HCl, 1.26; menadione (vit. K), 1.26; /I-carotene, 3.03; cr-tocopherol, 6.31; cyanocobalamine, 0.025; calciferol, 0.38; sodium-ascorbate (ascorbic acid), 630.92; folic acid, 0.25; choline chloride, 189.27. 2Mineral mix (mg/lOO g dry diet): KHPOI, 2.33; Ca,(PO,),, 3.18; MgS0,*7H20, 3.56; NaHP0,.2H20, 0.924.
(initial) /time (days), feed conversion ratio was computed as FCR= feed fed/ weight gain, and weight gain was calculated as WG= weight (final) -weight (initial) /time (days), each on a biweekly basis. Biweekly samples of 10 individuals from each of the four treatments were taken for histological analysis. The hepatopancreas was removed, fixed in Bouin’s solution for at least 24 h, processed in different grades of alcohol, cleared, and embedded in paraffin. Sections were cut at 5 pm with a rotary microtome and stained with haematoxylin and eosin (Humason, 1967). Results were analyzed using analysis of variance for a completely randomized design and Duncan’s multiple range test was used to separate means (Chang, 1972). RESULTS
A consistent trend of decreasing mean body weight was observed in prawns fed diets stored at increasing temperature (Table 2 ). Differences among diets
90
TABLE 2 Mean weight (g) of P. monodon juveniles fed diets stored at various temperatures (means of five replicates)’ Storage temperature (“C) 0
10 28-31 40 SEM
Time (weeks) 2
4
6*
a**
lo**
19.5” 18.5” 17.6” 18.2” 0.01
29.9” 35.5” 29.3” 25.3” 0.02
31.6” 34.5” 26.7’ 24.6b 0.47
37.1” 39.7” 24.3b 19.1’ 0.02
36.7” 38.5” 30.2b 19.9” 0.02
‘Treatment means with the same superscript are not significantly different within a given sampling period. *Significant at P-c 0.05; **significant at P-c 0.01. TABLE 3 Mean weight gain, specific growth, survival and feed conversion ratio (FCR) of P. monodon juveniles reared for 10 weeks on diets stored at various temperatures’ Storage temperature (“C)
Weight gain (8)
Specific growth (%)
Survival (%)
FCR (%)
0 10 28-31 40
36.9” 38.5” 30.2b 20.0”
8.88” 8.22” 7.27b 6.25’
76” 77.4” 67.gb 59.9”
8.9” 9.0” 10.5b 12.9
‘Treatment means with the same superscript are not significantly different at PC 0.05.
were not significant during the first 4 weeks of feeding. However, by the sixth week, the mean weight gain of prawns fed the diets stored at 2&J”-31 “C (26.7 g) and40”C (24.6 g ) was significantly less than for the other two treatments (P < 0.05 ). By week eight, prawns fed the diet stored at 40’ C (19.1 g ) weighed significantly less (PC 0.01) than those fed diets stored at 28”-31 ‘C (24.3 g) and at 0” or 10°C (38.7 g). The mean weight gain, specific growth, survival rate and feed conversion ratio for the various treatments after 10 weeks of rearing are shown in Table 3. Weight gain and specific growth rate were significantly higher (P < 0.01) for prawns fed diets stored at 0 o and 10’ C. Survival rate after 10 weeks of rearing was only 60% for prawns fed the diet stored at 40°C and feed conversion rate was higher for prawns fed the diet stored at the high temperatures (11.7% ) than for the prawns fed the diet stored at the low temperatures (9.0% ). Parallel to the growth response, significantly higher (P < 0.01) peroxide val-
91
0
l
0 w
0% AMBIENT 10°C 40%
2
4
6
6
IO
WEEKS
Fig. 1. Changes in peroxide value of diets stored at various temperatures.
Fig. 2. Section of the hepatopancreas of P. monodon fed a diet stored at low temperatures (1OT) showing well arranged hepatopancreatic tubules with tall columnar epithelial cells (arrow n) containing numerous secretory cells (B) and lipid vacuoles. Haematoxylin and eosin. ( x 400‘).
Fig. 3. Section of the hepatopancreas of P. monodon after 4 weeks of feeding a diet stored aItI high temperature (28 ’ -40 oC ) showing low cuboidal epithelial cells with decreased secretory cclI1s and lipid vacuoles (arrows). Necrotized epithelial cells became septic in the lumen (L) of the nepatopancreatic tubules. Haematoxylin and eosin. ( x 400).
Fig. 4. Section of the hepatopancreas of P. monodon after 10 weeks of feeding a diet stored aItshigh temperature (28”-40°C). Arrows point to disarranged hepatopancreatic cells with necr ‘CGzed epithelial cells in the lumen. Haematoxylin and eosin. ( x 400).
93
ues were observed in feeds stored at 28”-31 “C (9.3 meq/kg) and 40°C (10.9 meq/kg) compared to feeds stored at 0” or 10°C (3.0 meq/kg) starting with the fourth week of storage (Fig. 1) . In the hepatopancreas of prawn fed the diets stored at 0” and lO”C, the epithelial cells were tall and contained an abundance of secretory (B ) cells and R cells with numerous lipid vacuoles (Fig. 2). However, abnormal changes in the hepatopancreas were observed within 4 weeks for prawns fed diets stored at 28’ -31’ C and 40’ C. There was a reduction in the height of epithelial cells and an apparent decrease of B cells as well as lipid vacuoles (Fig. 3). The necrotized epithelium became septic in the luminar tubules of the hepatopancreatic cells. After 10 weeks of feeding the diets, the hepatopancreatic cells were disrupted and the epithelial cells were severely necrotized (Fig. 4). DISCUSSION
The growth of P. monodon juveniles fed diets stored at 28’ -31 oC and 40 ’ C became significantly reduced after 4 weeks of feeding. In addition, histopathological changes, including disrupted hepatopancreatic tubules, necrosis and epithelial detachment of the hepatopancreatic cells, started to appear at 4 weeks in the hepatopancreas of the animal. The increase in peroxide value could be attributed to oxidation of nutrients such as lipids and fatty acids, fat-soluble vitamins and vitamin C, as observed by Kulikov ( 1978) in his experiment on production of meal, oil and protein-vitamin preparations. According to Stuart et al. ( 1985)) feed stored at high temperature develops oxidative rancidity which affects growth and consumption in amphipods. In the present investigation, reduced growth and an apparent roughness of the exoskeleton were observed in P. monodon fed with diet stored at high temperatures (28’ -40 ’ C ) . The lower growth rates could be explained by the severe damage to the hepatopancreatic cells observed. According to Travis (1955) and Johnson (1980), the hepatopancreas is responsible for active metabolic reactions and also for storage of lipid used in cellular metabolism, moulting and reproduction. Gibson and Barker (1979) reported that the hepatopancress is responsible for the synthesis and secretion of digestive enzymes and subsequent uptake of nutrient materials. The hepatopancreas can be used as a monitoring organ for the nutritional value of diets in P. monodon (Vogt et al., 1985). Normally, a hepatopancreas contains four different types of cells in the epithelium. These are the B cells, R cells, F cells, and E cells (Johnson, 1980). In a normal hard-shelled prawn, B cells predominate (Baticados et al., 1987) and the presence of numerous B cells is characteristic of prawns that are actively feeding (Johnson, 1980). Necrosis of hepatopancreatic cells reduces the abundance of B cells and this may have led to impaired metabolism, causing a significant decrease in growth of P. monodon fed diet stored at high temperatures. Reduction in height of epi-
94
thelial cells of the hepatopancreas from tall columnar to cuboidal contributed to the malfunctioning of this organ. Lightner et al. (1982) also reported reduction in height of the proximal tubule epithelium of the hepatopancreas in marine shrimps (P. stybostris and P. uannamei) affected with aflatoxicosis. In order to decrease the rate of oxidation, the storage temperature should be reduced and storage conditions must be properly controlled (Kulikov, 1978). This is confirmed in the present study wherein quality deteriorated if feed was not stored at low temperature, as evidenced by the increasing peroxide value. According to Hardy (1980) the most effective prevention of oxidation is storage at low temperature; the oxidation rate can be further reduced with the removal of oxygen. Cho et al. (1985) reported that during storage, feed quality diminishes due to waste, chemical deterioration and infestation by insects or microorganisms. In the present study, waste from broken pellets and chemical deterioration occurred but no insects or microorganisms were observed. Based on these results, it is recommended that feeds should be stored for no more than 15 days during summer months when the temperature ranges from 28” to 31°C. Feed could be stored for up to 30-45 days during cooler months (10” -20’ C ). It is also suggested that a periodic check of peroxide levels should be done in order to detect the early stage of feed deterioration. ACKNOWLEDGEMENTS
The authors wish to thank Dr. Yvonne Chiu and Ms. Monina their advice and early correction of the manuscript.
Parazo
for
REFERENCES AOAC, 1975. Official Methods of Analysis, 12th edition. Association of Official Analytical Chemists, Washington, DC, 1094 pp. Baticados, M.C.L., Coloso, R.M. and Duremdez, R.C., 1987. Histopathology of the chronic softshell syndrome in the tiger prawn Penaeus monodon. Dis. Aquat. Org., 3: 13-28. Chang, L.C., 1972. The concept of statistics in connection with experimentation. ASPAC Food and Fertilizer Technology Center, Taipei City, Taiwan, Ext. Bull. No. 13, pp. 26-30. Cho, C.Y., Cowey, C.B. and Watanabe, T., 1985. Finfish nutrition in Asia: methodological approaches to research and development. IDRC, Ottawa, Ont., 154 pp. Chow, K.W., 1980. Storage problems of feedstuffs. In: Fish Feed Technology. FAO/UNDP Training Course, College of Fisheries, Univ. Washington, Seattle, WA, 9 October-15 December 1978. ADCP/REP/BO/ll, pp. 216-224. Gibson, R. and Barker, P., 1979. The decapod hepatopancreas. Oceanogr. Mar. Biol. Rev., 17: 285-346. Hardy, R., 1980. Fish lipid. In: J.J. Connel and staff of Torry Research Station (Editors), Advances in Fish Science and Technology. Torry Research Station, pp. 103- 111. Humason, G.L., 1967. Animal Tissue Technique, 2ndedition. W.H. Freeman, San Francisco, CA, 631 pp.
95 Johnson,
P.T., 1980. Histology of the Blue Crab, Callinectes sap&s.
Praeger, New York, NY, 440
PP.
Kulikov, P.I., 1978. Production of meal, oil and protein-vitamin preparations in the fishing industry. American Publishing Co. PVT. Ltd., Bombay, Calcutta, New York, NY, 253 pp. Lightner, D.V., Redman, R.M., Price, R.L. and Wiseman, M.O., 1982. Histopathology of aflatoxicosis in the marine shrimp Penaeus stylirostris and Penaeus vannamei. J. Invertebr. Pathol., 40: 279-291. Pascual, F.P., Coloso, S.M. and Tamse, C.T., 1983. Survival and some histological changes in Penaeus monodon Fabricius juveniles fed various carbohydrates. Aquaculture, 31: 169-180. Roberts, R.J., 1978. Fish Pathology. Bailliere Tindall, London, 318 pp. Rumsey, G.L., 1980. Stability of micro-ingredients in fish feeds. In: Fish Feed Technology. FAO/ UNDP Training Course, College of Fisheries, Univ. Washington, Seattle, WA, 9 October-15 December 1978. ADCP/REP/80/11, pp. 226-229. Stuart, V.E., Head, J.H. and Mann, K.H., 1985. Seasonal changes in the digestive enzyme levels of the amphipod Corophium volutator (Pallas) in relation to diet. J. Exp. Mar. Biol. Ecol., 88: 243-256. Travis, D.F., 1955. The molting cycle of the spiny lobster, Panulirus argus Latreille. II. Preecdysial histological and histochemical changes in the hepatopancreas and integumental tissues. Biol. Bull. (Woods Hole), 108: 88-112. Vogt, G., Starch, V., Quinitio, E.T. and Pascual, F.P., 1985. Midgut gland as monitor organ for the nutritional value of diets in P. monodon (Decapoda ) . Aquaculture, 48: I- 12.
87 -
Printed
in The Netherlands
Effect of Storage Temperature on the Quality of Diets for the Prawn, Penaeus monodon Fabricius MARGARITA
C. DE LA CRUZ, GREGORIA
ERAZO and MYRNA N. BAUTISTA
Aquaculture Department, Southeast Asian Fisheries Development Center, Tigbauan, Zloilo (The Philippines) (Accepted
14 October 1988)
ABSTRACT De la Cruz, M.C., Erazo, G. and Bautista, M.N., 1989. Effect of storage temperature of diets for the prawn, Penaeus monodon Fabricius. Aquaculture, 80: 87-95.
on the quality
The effect of storage temperature was evaluated on the basis of growth response of prawns fed for 10 weeks with diets stored at 0°C lO”C, 28”-31°C (ambient temperatures) and 40°C for a period of 10 weeks. Prawns were stocked at 15 pieces per 60-l oval tank supplied with water at 28°C and 32 ppt in a flow-through aerated system. There were five replicate tanks per treatment. Lowest weight gain (20 g) was observed for prawns fed the diet stored at 40°C and significantly higher growth response was observed as the storage temperature decreased (30.2 g at 28-31°C; 37.7 g at 0 ’ C and 10 ’ C ) . Body size was significantly (P < 0.05) affected by diet after 6 weeks of feeding and highly significantly (PC 0.01) after 8 weeks of culture. Peroxide values for diets exposed for 10 weeks to 28” -31 “C (12.8 meq/kg) and 40°C (15.0 meq/kg) were significantly higher than those exposed to 0” and 10°C (2.9 meq/kg). The highest survival rate (76%) and feed conversion (8.9%) were observed for prawns fed diets stored at low temperatures (0” or 1O’C). Severe necrosis of the hepatopancreatic cells was observed in P. monodon fed with diet stored at the high temperature.
INTRODUCTION
Most fish farmers do not realize the importance of proper feed storage. Often, they are more concerned with seasonal fluctuations in availability and price of feed, and resort to bulk purchases which are stored for long periods. During prolonged and improper storage, adverse physical conditions (moisture, heat, light ) and microorganisms (molds, bacteria, yeast) may cause deterioration of feed quality. The resulting decrease in palatability and nutritive value, including deterioration of amino acids, vitamins and fat (Chow, 1980), can lead to economic losses. The rate of oxidation of fat is greatly influenced by storage temperature and a rise of 10’ C approximately doubles this rate (Kulikov, 1978). Oxidation of fat gives rise to a feedstuff of lower biological energy value (Rum-
0044-8486/89/$03.50
0 1989 Elsevier Science Publishers
B.V.
88
sey, 1980) that may cause a reduction in growth of the animal (Stuart et al., 1985 ) . The use of deteriorated feeds also can result in increased disease outbreaks including one known as “lipoid liver degeneration” (Roberts, 1978). Travis (1955) considered the hepatopancreas as a storage organ of organic and mineral reserves. It is also a monitoring organ for the nutritional value of diets for P. monodon (Vogt et al., 1985). Since several authors (Johnson, 1980; Pascual et al., 1983; Vogt et al., 1985; Baticados et al., 1987) have reported the importance of the hepatopancreas in evaluating the nutritional value of the diet, it is imperative to include examination of this organ in assessing the quality of diets. The study reported here was conducted to determine the degree of rancidity of diets, and the growth, survival, and histopathological effects on the hepatopancreas of P. monodon juveniles fed diet stored at varying temperatures. MATERIALS AND METHODS
A practical diet for prawns (Table 1) was prepared by mixing all the dry ingredients in a Hobart mixer for 10 min. The oil was gradually added to the mixture and mixed for another 5 min and then a sufficient quantity of warm water to give a smooth consistency was added to the oil mixture and blended for another 5 min. A Hobart meat grinder with a 2-mm die was used for diet extrusion. The extruded moist pellets were broken into small pieces, divided into four batches, and stored in plastic bags at four different temperatures: 0’) 10’) 28-31’) and 40’ C for a period of 10 weeks. The proximate analysis of the diet was performed using standard methods ( AOAC, 1975 ) . Three hundred and sixty P. monodon juveniles with a mean weight of 1.5 g and standard deviation of 0.5 g were obtained from SEAFDEC ponds at Leganes, Iloilo, The Philippines, and acclimatized for 1 week under laboratory conditions while being fed crustacean pellets. The prawns were randomly distributed to 20,60-l tanks provided with continuous aeration and seawater filtered through sand and gravel at a rate of 0.8-1.0 l/min. Temperature and salinity ranged from 28” to 29°C and 32 to 34 ppt, respectively. Prawns were stocked at 15 pieces per tank, with five replicate tanks per treatment. Five PVC pipes, about 6 in (15 cm) long and 2 in (5 cm) in diameter were placed in each tank to provide cover and prevent cannibalism. Feeding of the test diet was done twice a day at 10% of body weight for the first 2 weeks, after which the feed was reduced gradually to 2% at 10 weeks. The amount fed was adjusted after biweekly sampling of prawns when the prawns were weighed individually. Faeces and uneaten feed were siphoned out every morning. Mortalities and molt stages were noted daily. The oval tanks were thoroughly cleaned when the prawns were removed for weighing every 2 weeks. Specific growth rate was computed as SGR = log weight (final ) - log weight
89 TABLE 1 Composition of experimental diet Ingredients
g/100 g dry diet
Fish meal Squid meal Shrimp head meal Soybean meal (defatted) Rice bran Bread flour Cod liver oil Soybean lecithin Vitamin mix’ Mineral mix’ Cholesterol
15 15 15 15 14 15 5 2 1.5 1.5 1.0
Nutrients
Proximate composition (% as feed)
Moisture Crude protein Crude fat Crude fiber NFE Ash
7.15 45.02 17.6 4.20 24.51 9.11
IVitamin mix (mg/lOO g dry diet); p-aminobenzoic acid, 3.16; biotin, 0.10; inositol, 126.18; nicotinic acid, 12.62; calcium pantothenate, 18.93; pyridoxine HCl, 3.17; riboflavin, 2.57; thiamine HCl, 1.26; menadione (vit. K), 1.26; /I-carotene, 3.03; cr-tocopherol, 6.31; cyanocobalamine, 0.025; calciferol, 0.38; sodium-ascorbate (ascorbic acid), 630.92; folic acid, 0.25; choline chloride, 189.27. 2Mineral mix (mg/lOO g dry diet): KHPOI, 2.33; Ca,(PO,),, 3.18; MgS0,*7H20, 3.56; NaHP0,.2H20, 0.924.
(initial) /time (days), feed conversion ratio was computed as FCR= feed fed/ weight gain, and weight gain was calculated as WG= weight (final) -weight (initial) /time (days), each on a biweekly basis. Biweekly samples of 10 individuals from each of the four treatments were taken for histological analysis. The hepatopancreas was removed, fixed in Bouin’s solution for at least 24 h, processed in different grades of alcohol, cleared, and embedded in paraffin. Sections were cut at 5 pm with a rotary microtome and stained with haematoxylin and eosin (Humason, 1967). Results were analyzed using analysis of variance for a completely randomized design and Duncan’s multiple range test was used to separate means (Chang, 1972). RESULTS
A consistent trend of decreasing mean body weight was observed in prawns fed diets stored at increasing temperature (Table 2 ). Differences among diets
90
TABLE 2 Mean weight (g) of P. monodon juveniles fed diets stored at various temperatures (means of five replicates)’ Storage temperature (“C) 0
10 28-31 40 SEM
Time (weeks) 2
4
6*
a**
lo**
19.5” 18.5” 17.6” 18.2” 0.01
29.9” 35.5” 29.3” 25.3” 0.02
31.6” 34.5” 26.7’ 24.6b 0.47
37.1” 39.7” 24.3b 19.1’ 0.02
36.7” 38.5” 30.2b 19.9” 0.02
‘Treatment means with the same superscript are not significantly different within a given sampling period. *Significant at P-c 0.05; **significant at P-c 0.01. TABLE 3 Mean weight gain, specific growth, survival and feed conversion ratio (FCR) of P. monodon juveniles reared for 10 weeks on diets stored at various temperatures’ Storage temperature (“C)
Weight gain (8)
Specific growth (%)
Survival (%)
FCR (%)
0 10 28-31 40
36.9” 38.5” 30.2b 20.0”
8.88” 8.22” 7.27b 6.25’
76” 77.4” 67.gb 59.9”
8.9” 9.0” 10.5b 12.9
‘Treatment means with the same superscript are not significantly different at PC 0.05.
were not significant during the first 4 weeks of feeding. However, by the sixth week, the mean weight gain of prawns fed the diets stored at 2&J”-31 “C (26.7 g) and40”C (24.6 g ) was significantly less than for the other two treatments (P < 0.05 ). By week eight, prawns fed the diet stored at 40’ C (19.1 g ) weighed significantly less (PC 0.01) than those fed diets stored at 28”-31 ‘C (24.3 g) and at 0” or 10°C (38.7 g). The mean weight gain, specific growth, survival rate and feed conversion ratio for the various treatments after 10 weeks of rearing are shown in Table 3. Weight gain and specific growth rate were significantly higher (P < 0.01) for prawns fed diets stored at 0 o and 10’ C. Survival rate after 10 weeks of rearing was only 60% for prawns fed the diet stored at 40°C and feed conversion rate was higher for prawns fed the diet stored at the high temperatures (11.7% ) than for the prawns fed the diet stored at the low temperatures (9.0% ). Parallel to the growth response, significantly higher (P < 0.01) peroxide val-
91
0
l
0 w
0% AMBIENT 10°C 40%
2
4
6
6
IO
WEEKS
Fig. 1. Changes in peroxide value of diets stored at various temperatures.
Fig. 2. Section of the hepatopancreas of P. monodon fed a diet stored at low temperatures (1OT) showing well arranged hepatopancreatic tubules with tall columnar epithelial cells (arrow n) containing numerous secretory cells (B) and lipid vacuoles. Haematoxylin and eosin. ( x 400‘).
Fig. 3. Section of the hepatopancreas of P. monodon after 4 weeks of feeding a diet stored aItI high temperature (28 ’ -40 oC ) showing low cuboidal epithelial cells with decreased secretory cclI1s and lipid vacuoles (arrows). Necrotized epithelial cells became septic in the lumen (L) of the nepatopancreatic tubules. Haematoxylin and eosin. ( x 400).
Fig. 4. Section of the hepatopancreas of P. monodon after 10 weeks of feeding a diet stored aItshigh temperature (28”-40°C). Arrows point to disarranged hepatopancreatic cells with necr ‘CGzed epithelial cells in the lumen. Haematoxylin and eosin. ( x 400).
93
ues were observed in feeds stored at 28”-31 “C (9.3 meq/kg) and 40°C (10.9 meq/kg) compared to feeds stored at 0” or 10°C (3.0 meq/kg) starting with the fourth week of storage (Fig. 1) . In the hepatopancreas of prawn fed the diets stored at 0” and lO”C, the epithelial cells were tall and contained an abundance of secretory (B ) cells and R cells with numerous lipid vacuoles (Fig. 2). However, abnormal changes in the hepatopancreas were observed within 4 weeks for prawns fed diets stored at 28’ -31’ C and 40’ C. There was a reduction in the height of epithelial cells and an apparent decrease of B cells as well as lipid vacuoles (Fig. 3). The necrotized epithelium became septic in the luminar tubules of the hepatopancreatic cells. After 10 weeks of feeding the diets, the hepatopancreatic cells were disrupted and the epithelial cells were severely necrotized (Fig. 4). DISCUSSION
The growth of P. monodon juveniles fed diets stored at 28’ -31 oC and 40 ’ C became significantly reduced after 4 weeks of feeding. In addition, histopathological changes, including disrupted hepatopancreatic tubules, necrosis and epithelial detachment of the hepatopancreatic cells, started to appear at 4 weeks in the hepatopancreas of the animal. The increase in peroxide value could be attributed to oxidation of nutrients such as lipids and fatty acids, fat-soluble vitamins and vitamin C, as observed by Kulikov ( 1978) in his experiment on production of meal, oil and protein-vitamin preparations. According to Stuart et al. ( 1985)) feed stored at high temperature develops oxidative rancidity which affects growth and consumption in amphipods. In the present investigation, reduced growth and an apparent roughness of the exoskeleton were observed in P. monodon fed with diet stored at high temperatures (28’ -40 ’ C ) . The lower growth rates could be explained by the severe damage to the hepatopancreatic cells observed. According to Travis (1955) and Johnson (1980), the hepatopancreas is responsible for active metabolic reactions and also for storage of lipid used in cellular metabolism, moulting and reproduction. Gibson and Barker (1979) reported that the hepatopancress is responsible for the synthesis and secretion of digestive enzymes and subsequent uptake of nutrient materials. The hepatopancreas can be used as a monitoring organ for the nutritional value of diets in P. monodon (Vogt et al., 1985). Normally, a hepatopancreas contains four different types of cells in the epithelium. These are the B cells, R cells, F cells, and E cells (Johnson, 1980). In a normal hard-shelled prawn, B cells predominate (Baticados et al., 1987) and the presence of numerous B cells is characteristic of prawns that are actively feeding (Johnson, 1980). Necrosis of hepatopancreatic cells reduces the abundance of B cells and this may have led to impaired metabolism, causing a significant decrease in growth of P. monodon fed diet stored at high temperatures. Reduction in height of epi-
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thelial cells of the hepatopancreas from tall columnar to cuboidal contributed to the malfunctioning of this organ. Lightner et al. (1982) also reported reduction in height of the proximal tubule epithelium of the hepatopancreas in marine shrimps (P. stybostris and P. uannamei) affected with aflatoxicosis. In order to decrease the rate of oxidation, the storage temperature should be reduced and storage conditions must be properly controlled (Kulikov, 1978). This is confirmed in the present study wherein quality deteriorated if feed was not stored at low temperature, as evidenced by the increasing peroxide value. According to Hardy (1980) the most effective prevention of oxidation is storage at low temperature; the oxidation rate can be further reduced with the removal of oxygen. Cho et al. (1985) reported that during storage, feed quality diminishes due to waste, chemical deterioration and infestation by insects or microorganisms. In the present study, waste from broken pellets and chemical deterioration occurred but no insects or microorganisms were observed. Based on these results, it is recommended that feeds should be stored for no more than 15 days during summer months when the temperature ranges from 28” to 31°C. Feed could be stored for up to 30-45 days during cooler months (10” -20’ C ). It is also suggested that a periodic check of peroxide levels should be done in order to detect the early stage of feed deterioration. ACKNOWLEDGEMENTS
The authors wish to thank Dr. Yvonne Chiu and Ms. Monina their advice and early correction of the manuscript.
Parazo
for
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