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Lipid and fatty acid composition of milkfish (Chanos chanos Forsskal) grown in freshwater and seawater
Aquaculture, IO4 ( I992 ) 79-89 Elsevier Science Publishers B.V., Amsterdam
79
I.G. Borlongan and L.V. Benitez Aquaculture Department, SEAFDEC, Iloilo City, Philippines
(Accepted 5 September 1991)
APSTRACT
Borlongan, I.G. and Benitez, L.V., 1992. Lipid and fatty acid composition of milktish ( Chanos chanos Forsskal) grown in freshwater and seawater. Aquaculture, 104: 79-89. The lipid and fatty acid compositions of the various organs of milkfish fed with an invariant diet and reared in seawater (SW) and freshwater (FW) were determined using column chromatography and gas chromatography. Phospholipid content of the gills, kidney, liver, intestines and depot fat was higher in SW than in FW while the organs from fish in FW had higher contents of neutral lipid. Fatty acid patterns of total lipids in the liver, intestines and depot fat of mi!kf ;h reared in FW and SW were similar. There were marked differences in fatty acid patterns of gills.zikd kidn:y. The proportions of saturated to unsaturated fatty acids in gills and kidney were lower in SW than in FW. Likewise, the ratio of n-3 to n-6 fatty acids and total polyunsaturated fatty acids ( PUFAs) [Jfgills and kidney were higher in SW than in FW. The fatty acid patterus of the phospholipid fractionr, showed that SW-reared milkfish have higher total PUFAs, especially of the n-3 fatt;) acids, than the FW-reared milkfish not only in gills and kidney but in all organs examined. The differences in lipid ard fatty acid composition reflect a physiological response to the salinity in which milkfish were reared.
INTRODUCTION
The growing interest in farming milkfish both in brackishwater ponds and freshwater pens requires further insight into fish biochemistry and physiology during changes in salinity. The milkfish ( Charmschanos Forsskal ) is a euryMine species. It can thrive in natural waters of salinity ranging from 0 to 100 ppt (Crear, 1980; Dalagan, 1980; Pullin, 198 1). It is most comtionl~ cultured in brackishwater ponds where its ability to withstand wide and rapid changes in salinity is well-known. Lipids and fatty acids pray an important role in membrane biochemistry Correspondence to: LG. Borlongan, Aquaculture Department, Southeast Asian Fisheries velopment Center, P.O. Box 256, Iloilo City, Philippines.
0044~8486/92/$05.00
0 1992 Elsevier Science Publidrers B.V. All rights reserved.
De-
80
LG. BORLONGAN AND L.V. BENlTEi!
and therefore have direct impact on such membrane-mediated processes as osmoregulation, reproduction, nutrient assimilation and transport. Several studies have shown the usefulness of tissue fatty acid profiles in predicting the essential fatty acid needs of cultured fish (Castell, 1979 ). The fatty acid profiles of marine fish eggs (Dendrinos and Thorpe, 1987 ), larvae (Dendrinos and Thorpe, 1987; Fraser et al., 1987; Teshima et al., 1987 ), and juveniles (Ring0 and Nilsen, 1987; Ostrowski and Divakaran, 1989) have been used to predict the dietary requirements of fishes. Several studies have also shown that. the fatty acid composition of the diet influences that oi the fish (Cowey and Sargent, 1977; Watanabe, 1982; Stickney and Hardy, 1989). In additi,n to dietary considerations, the fatty acid composition of the fish is affected by environmental factors. The differences may be related to a specific requirement in fish enabling them to adapt physiologically to the environment. Although there are probably fish species with specific essential fatty acid (EFA) requirements, the influence of environmental factors is so great that the EFA requirements of a single species are changed by variation in temperature and salinity (CastelI, 1979). Studies have shown that salinity of water affects the fatty acid composition, especially the PUFA levels of fish. The ratio of n-3 to n-6 fatty acids is much lower in fish that thrive in FW than in a SW environment. These studies showed that the ratio of n-3 to n-6 fatty acids ranges from 1.7 to 3.5 and from 7.5 to 19.5 for freshwater and marine species respectively (Gruger et al., 1964; Ackman, 1967; Standsby, 1967). Whether these patterns are due to the effect of salinity alone or to differences in the natural foods present in these environments needs to be determined. The purpose of this work is to determine the lipid composition and fatty acid profile of the various organs of milkfish fed the same diet but cultured in freshwater and seawater. This information might prove both theoretically im-
portant to explain adaptation of euryhaline fish to salinity changes, and practically important as an aid to diet formulation in aquaculture. MATERIALS AND METHODS
Fish, salinityacdimation and experimentalrurt Milkfish weighing about 150-l 75 g were collected from fishponds with a salinity of 3 1 ppt and were transported to the laboratory. The fish were stocked in four canvas tanks filled with 10 tons of seawater. Each tank was stocked with 120 fishes. The fish were acclimated to laboratory conditions for 1 week and trained to accept pelleted feeds. At the end of this period, the salinity in two of the culture tanks was gradually reduced at the rate of 2 ppt per day by the introduction of freshwater until the salinity was about 0 ppt (FW ). The salinity of the two other tanks was maintained at full strength seawater (32 PP4 SW.
Both groups of fish were fed twice daily with the same commercial diet
LIPID AND FATTY ACID COMPOSTION
OF MILKFISH
81
(40% crude protein) at the raLe of 5% biomass per day. After 47 days from stocking, the t,wu groups of fish were fully acclimate to the salinity in their respective culture tanks and fully adapted to pelleted feeds. The fish were reared for another 60 days beyond full acclimation, for a total of 107 days. The water in both tanks was supplied by a flow-through system and supplemental aeration was provided. Selected physico-chemical parameters of the culture water, namely pH, dissolved oxygen, salinity, temperature, ammonia, nitrite and phosphate levels were determined thrice weekly using standard methods (APHA, 1976). Lipid analysis Samples of 30 fish each were collected at the start, at day 30, and at day 60 after full acclimation from- each tank of fish reared in freshwater (FW) and seawater (SW ) . Individual weight of fish was recorded and the fish were then dissected to isolate five different organs, namely. gills, kidney, liver, intestines and depot fat. Identical organs from each batch of 30 fish per tank were pooled and total lipid content of each organ was determined by the method ( 1959 ). Duplicate analyses were carried out on each pool. The total lipid was further resolved into neutral lipid, phospholipid and glycolipid fractions by silicic acid column chromatography using the method at 0.05% was of Moerck and Ball ( 1973 ) . utylated hydroxytolu&e ( tal lipid fracadded to all solvent systems prevent lipid oxidation. tion was converted into fatty acid methyl esters by saponification with 0.5 N and transesterified with boron trifluoride methanol remethanolic Na agent (Metcal al., 1966 ). The fatty acid methyl esters were analyzed on a Shimadzu CC-4C gas chromatograph equipped with a flame ionization detector and using a stainless steel 15% diethyleneglycol succinate mosorb W support. he column temperature was set at 180°C and fatty acids were identified by comparison with known standards. Ch dards of polyunsa.turated fatty acid methyl esters (No. 7015) were purchased from Supelco, USA. All other reagents we ical grade. Statistical analysis One-way analysis of variance and uncan’s multiple range test were use for statistical comparison of results between organs and Student’s t-test for the comparison of results between treatments (freshwater versus seawater) (Snedecor and Cochran, 1980). Differences were considered significant at Pc 0.05 level. RESULTS
Physics-chemical parame,“ers The analyzed physico-chemical parameters of the culture water were within tolerable limits. Dissolved oxygen ranged from 4.4 to 7.4 ppm in the seawater
LG. BORLONGAN AND L.V. BENITEZ
82
tank and 5.5 to 9.1 ppm in the freshwater tank. The concentrations of nitrite and ammonia ranged from zero to negligible values for both fresh and seawater tanks for the duration of the feeding trial. Temperature ranged from 26 to 32 “C and pH from 7.6 to 8.4 for both fresh and seawater ta.nks. The seawater tank had a salinity of about 27 to 32 ppt and the freshwater ‘rtinkhad zero or negligible salinity. Effect of salinity on lipid composition
Total lipid content expressed as percentage of wet tissue weight of the various organs of milkfish reared in freshwater and seawater is shown in Table 1. In the initial pond samples, the highest lipid content was observed in the gills. After the 30-day culture period, the lipid content of the gills and kidney in both FW and SW reared fish were comparable with the initial samples. However, there was a significant increase in the total lipid content in the depot fat and liver but a significant decrease in the intestines. At the end of the 60-day culture period, significant increase in the total lipid content was observed in the depot fat, liver and gills but there was a significant decrease in the kidney and intestines for both the FW and SW reared fish. At both day 30 and 60, there was no significant difference in the total lipid content in all organs between FW and SW reared fishes. Results of the fractionation (Tables 2-4) of the total lipids showed that of the three lipid classes, the neutral lipid (65-78%) was predominant in all organs of ftsh reared in both freshwater and seawater. This was followed by phospholipids ( 13-26%) and glycolipids ( 5- 11%). No significant differences were found in the % neutral lipid content of the depot fat and liver between seawater and freshwater reared fish (Table 2). However, the gills, kidney and intestines from fish reared in freshwater had significantly higher TABLE I Total lipid content of various organs of milkflsh reared in freshwater and seawater Organs
Total lipids’*2 (g/ 100 g wet weight tissue) Initial pond samples
Day 30 FW
Gills Kidney Liver Intestines Depot fat
7.5 kO.9’ 4.3 + 0.3” 4.3%0.5” 3.6+0.7a 5.7 -i-0.6b
8.6f0.3’ 5.3f0Sb 6.0 + 0.3b l.4+0.8a Il.O& 1.5d
Day 60 SW
FW
SW
8.7f0.4C 9.6&0.3b 9.7+0.5b 4.7 f 0.4b 1.1kO.3” 1.9* 1.0” 5.7+0.5b 10.9f 1.3b 13.0 + 2.4’ 2.2 + o.7a 1.1f0.5” I .4 + 0.8” 9.82 1.5’ 16.01t 1.3’ 14.6+ 1.6d .-_ ‘Means not sharing a common superscript in each column are significantly different at P< 0.05. “No significant differences in all organs between FW aud SW reared milk&h at PC 0.05.
LIPID AND FATTY ACID COMPOSITION OF MILKFISH
83
Neutral lipid content of various organs of milktish reared in freshwater and seawater Organs
Neutral lipids’ (% of total lipids) Initial pond samples
Gills Kidney Liver Intestines Depot fat
66.5 * 0.3” 66.7kO.2” 66.4 + 0.4a 67.7 f 0.5b 72.5 k 0.6’
Day 30
77.9+0.3e** 75 I +04d** 73:4 7 1:570.6 ? 0.3a++ 72.8 + 0.3b*“s
Day 60 SW
FW
SW
66.1 I!I 0.3b 68.1+0.7’ 70.9 f 0.7” 65.0&0.8” 72.1 kO.9’
75.2 +0.3d** 72.3 f 0.4’~” 7 1.6 + 0.5b*“S 69.3 f 0.7”*” 72.3 + 0.9’.“”
65.9 + 0.6b 64.7 +0.8” 71.2f0.5d 66.5 I!I0.9’ 71.6f0.7’
‘Means not sharing a common superscript in each column are significantly different at P-c 0.05. *Highly significant; “’ no significant differences between FW axi SW reared fish at P~0.05. TABLE 3 Phospholipid content of various organs of milkfish reared in freshwater and seawater Organs
Phospholipids’ ( W of total lipids) Initial pond samples
Gills Kidney Liver intestines Depot fat
23.1 t-0.1’ 22.6 ?I0.3” 25.9 + 0.4* 21.7tO.l” 22.4kO.l”
Day 30
Day 60
FW
SW
FW
SW
13.2-t 0.4’*** 14.0+ 0.4b*L* 18.1!I 0.4’** 18.4f 0.5c**+ 20.4 It 0.3d**
24.910.5’ 22.2+0.5b 20.9 + 0.3’ 25.7 etlO.Sd 22.5 kO.5”
16.3 + 0.48*** 18.1? 0.4b*+* 19.7 -t 0.4’,* 20.0 I?I0.2’,* 20.8 Z!I OS”*’
25.1 kO.3’ 25.8+0.5’ 20.7 III0.5” 23.0+0.5” ZE.7 II 0.4”
‘Means not sharing a common superscript in each column are significantly different at P-z 0.05. **Highly significant; + significant differences between FW and SW reared milkfish at PC 0.05.
percentage contents of neutral lipids than those of fish reared in seawater. The gills appeared to have the highest content of neutral lipids for the freshwater samples while the depot fat and liver had the highest level of neutral li the seawater samples. Milkfish reared in seawater had a significantly higher content of phosp lipids compared to those reared in fres initial fish samples obtained from the fi to that of the seaw to the samples rear a level of 13 to 2 1% for 21 to 26% in organs o
84 TABLE
LG. BCRLONGAN
AND L.V. BENITEZ
4
Glycoiipid content of various organs of milkfish reared in freshwater and seawater Organs
Glycolipids’*’ (96of total lipids) Initial pond sampies
Gills Kidney Liver Inteaines Depot fat
10.4 +_0.4’ 10.7fO.l’ 7.7+0.1b 10.6+0.7’ 5. I kO.4”
Day 60
Day 30 FW
SW
FW
SW
8.9+_0.5b io.9+ 1.F 8.5 fOSb I l.Of 1.7’ 6.9+ 1.3;’
9.010.5’ 9.7 + I .O’ 8.2 + 0.4b 9.3 +osc 5,4+ 1.5”
8.4 + 0.6b 9.6 + 0.5b 8.8 + 0.6b 10.7+0.8’ 6.9?0.5”
8.9 + 0.4b 9.5&0Sb 8.1 kO.7” 10.5?.0.4’ 5.7 -t 1.3”
‘Means not sharing a common superscript in each column are significantly different at P-z 0.05. ‘No significant differences in all organs between FW and SW reared fish at PC 0.05.
those reared in freshwater. The gills had lowest levels of phospholipid for fish reared in freshwater. Comparison of the glycolipid content of the various organs of milkfish reared in freshwater and seawater showed no significant differences (Table 4). In both groups of fish, significantly higher glycolipid content was observed in the intestines, kidney and gills (the organs associated with high numbers of epithelial cells), while the depot fat had the lowest glycolipid content.
omparison of fatty acid patterns of the total lipids from various organs of fishes reared in freshwater and seawater showed similar patterns in the liver, intestines and depot fat for both the Day 30 and Day 60 samples. However, marked differences were noted in the fatty acid patterns of the gills and kidneys, particularly for the Day 60 samples (Table 5 ). The gills from seawaterreared fishes contained large quantities of unsaturated fatty acids (72.5%) and much lower quantities of saturated fatty acids (27.5%). The opposite was observed in the freshwater samples where the gills contained larger quantities of saturated fatty acids (84.9%) than unsatu fatty acids ( 15.0%). The gills of SW-reared fish contained higher pert es of 16: 1 and 18: 1 FAs than the R&reared fish. In the kidney, the predominance of saturated fatty acids over unsaturated fatty acids in the freshwater samples was noted. Unlike the hills: this same trend was observed for the seawater samples. In both gills and kidney, the ratio of (n-3) to (n-6) fatty acids and the polyunsaturated fatty acid (PUFA) content were h in the seawater than in freshwater-reared fish. For both the FW and S red fish the predominant saturated FA in all organs was stearic ( 18 : 0) acid.
__
7.98* 1.49 5.36* 76.15 23.84* 9.47* 3.19
3.98 2.35 1.69 85.69* 14.29 6.33 5.99* 6.50 2.37 2.74 76.46 23.54 8.87 3.25
5.90 7.49 4.46 13.14 58.61* 1.02 1.11* 2.08 0.50 0.41 0.84 4.30 0.01 0.03 0.09
Intestines
6.40 1.67 3.83 78.88 21.12 8.07 3.73
11.86 8.50 3.48 10.98 55.04 0.66 0.42 1.80 1.34 0.42 0.83 4.51 0.07 0.06 0.03
SW
2.80 1.42 1.97 70.33 29.66 4.22 2.37
5.32 5.78 10.27* 10.72 48.96 14.42* 0.39 0.63 0.23 0.18 0.85 1.88 0.07 0.09 0.20
FW
Depot fat
2.07 1.45 1.43 78.66 21.35 3.52 3.68
5.81 lO.28* 2.92 16.57* 59.65 1.02 0.50 0.55 0.22 0.20 0.75 1.48 0.02 0.02 0.02
SW
4.05 1.66 2.44 80.82 19.16 5.71 4.22
1.60 4.06 10.34 12.57* 64.82 0.48 1.09 0.55 0.09 0.25 0.32 3.00 0.31* 0.07 0.43
Liver
7.05* 2.10 3.36 83.36 16.63 9.15’ 5.01
4.61 8.20’ 10.02 7.18 60.53 0.14 1.45 0.71 0.09 0.22 0.43 6.28* 0.07 0.03 0.03
SW
-
_.-_
-”
-
I
_
-
.
. -
‘Day 60 samples. *Composition expressed as weight % of total lipids. Values are me&s of four replicate znalyw-YS:r!ndard deviations are omitted for clarity ofpresentation but normally were c 5%. *Highly significant differences between FW and SW reared milkf:sh at PC 0.05 (Student’s r-test ).
Total satd FAs Total unsatd FAs Total PUFAs Satd/unsatd
(n-3) (n-9) (n-6) (n-6) (n-3) (n-9) (n-3) (n-3)
1.64 1.00 1.64 84.98+ 15.01 2.64 5.66
18:3 2O:l 20:3 20:4 20:s 22:1 22: 5 22:6
Total (n-3) I-As Total (n-6 ) FAs n-3/n-6 FAs
4.46 15.79 19.87* 12.72* 36.03 0.49 0.40 3.34* 1.09* 0.33 0.79 4.56* 0.07 0.04 0.04
3.24 14.44 3.90 6.76 64.11* 0.86 1.70* 1.37 0.17 0.24 0.41 2.43 0.17 0.03 0.15
0.14 2.06 0.71 42.59* 24.57 20.92* 1.16* 0.48 0.56 0.17 0.67 0.23 0.33* 1.88* 3.51* 6.10* 2.00 3.05* 27.48 72.50* 8.10* 0.36
2.00 14.84” 5.88* 11.85 62.26* 0.37 0.38 0.68 0.13 0.12 0.50 0.85 0.02 0.04 0.07
FW
SW
FW
SW
Kidney
Gills
Percentage composition*
12:O 14:o 16:O 16:l (n-7) 18:O 18:l (n-9) 18:2 (n-6)
Fatty acids
Fatty acid composition of the total lipids from various organs of milkfish reared in freshwater and seawater’
TABLE 5
0.79
21.29 23.57 10.83 30.43* 0.49 4.06* 0.49 0.32 0.4 1 0.99 0.35 3.22* 1.39* 9.66* 1.22 7.92* 34.28 6j.?2* 10.88*
0.52
0.88
1.48
0.23 1.84 20.86 21.30 21.11* 24.25* 0.43 1.53 1.48 0.78 0.65 0.30 1.98. 2.46 0.80 5.09 1.86 2.74 44.04 55.96 6.95
1.01 5.22 25.94 25.19 14.50 20.98 1.05 1.39 0.26 0.29 0.19 1.07 0.87 1.89 0.15 4.50 1.53 2.94 46.67* 53.33 6.03
0.68
FW
FW
SW
Kidney
Gills
Percentage composition*
0.83
0.63 6.44” 24.79 24.22 13.51 14.87 1.11 1.51 3.54* 0.82 0.22 0.51 1.51 3.92* 2.40* 8.34* 2.15 3.88” 45.37 54.63 10.49*
SW
13.26* 1.97 6.73* 41.37 58.63* 15.23* 0.71
7.06 2.14 3.30 50.78* 49.22 9.20 1.03
0.83 0.65 2.97 1.78
1.27*
0.79 0.85 30.66 25.02* 9.07 17.24 0.91 2.51* 0.37 0.52 0.54 5.38* 0.77 2.54 3.19*
SW
0.54 1.29 40.72* 17.62 8.23 17.80 0.72 1.48 3.95* 0.15
FW
Intestines
SW 0.18 0.12 4:.11* 19.03 3.97 12.77 0.62 1.06 5.55* 0.93 0.25 1.13* 0.67 ?.23* 5.38* 14.80* 1.80 8.22* 45.38 54.62 16.60* 0.83
FW 0.06 0.16 29.94 34.98* 10.73* 17.15 0.85 1.60 0.19 0.20 0.13 0.22 0.29 3.20 0 30 5.32 1.18 4.51 40.89 59.11 6.50 0.60
0.85 3.01 11.71 26.39 23.68* 26.14* 1.os* 2.54* 1.18* 0.42 0.78 2.61 0.03 0.05 0.07 5.27* 2.28* 2.31 38.7 1 61.29* 7.55* 0.63
0.91 0.48 35.42* 29.26 10.95 17.74 0.34 1.32 0.36 0.29 0.72 2.07 0.02 0.04 0.08 3.51 1.35 2.60 47.76* 52.24 4.86 0.9 1
Liver SW
FW
Depot fat ---
. . .. .
. . .. .
. __-
_ ._
_ ,_
_
as
,
_,
I
--
_
.._
‘Day 60 samples. ‘Composition expressed as weight Z of total phospholipids. Values are means of four replicate analyses. Standard deviations are omitted for clarity of presentation but normally were < 5%. *Highly significant differences between FW and SW reared milktish PC 0.05 (Student’s t-test ).
Satd/unsatd
12:0 14:o 16:0 16:1 (n-7) 18:O 18:l (n-91 t8:2 (n-6) 18:3 (n-3) 2O:l (n-9) 20:3 (n-6) 20:4 (n-6) 20: 5 (n-3) 22: 1 (n-9) 22:5 (n-3) 22:6 (n-3) Total (n-3) FAs Total (n-6) FAs n-3/n-6 FAs Total satd FAs Total tmsatd FAs Total PLJFAs
Fatry acids
Fatty acid composition of the phospholipids from various organs of milkfish reared in freshwater arnd seawater’
TABLE 6
8 g z $ g r .< g z =i A
5 $ F
_
_.
LlPlD AND FATTY ACID COMPOSLTION OF MILKFISH
87
Effect of stalinity on the fatty acid composition of phosphdipid frmions While salinity affected only the fatty acid patterns of the total lipids gills and kidney, it affected the fatty acid patterns of “ihephospholipid fractions of all organs examined (Table 6). There was a significantly higher centage of total PUPAS in all organs for the SW-reared fish compared to reared fish. The phospholipids from organs of SW-reared fish contained h percentages of n-3 fatty acids than FW-reared fish. Consequently, the ratios of n-3 to n-6 fatty acids of phospholipids from SW-reared fish were also higher. DlSCIJSSION
NO significant differences in the percentage total lipid content were observed in all organs of milkfish reared in FW and SW, indicating that salinity did not affect fat deposition in terms of amount of total lipid stored. HOWever, the composition of the different lipid classes ( phospholipid, neutral lipid and glycolipid) and fatty acids of milkfish changed with the salinity of the rearing water. The differences in the lipid and fatty acid composition in response to salinity were expressed principally in the gills, ki neys and intestines of milkfish. Phospholipid levels of gills, kidneys and intestines were 1of neutral lipids were higher in fishes reared in SW than in FW. Nigher 1 noted in those organs offish reared in FW than in Salinity significantly affected the fatty acid composition of the phospholipid fractions of all organs, while in the total lipids differences in fatty acid patterns were most pronounced in the gills and kidneys. The levels of unsaturated I%, ratio of (n-3) to (n-6) PAS and levels of PUFAs were higher in than in FW-reared fish. The change in lipid composition and fatty acid erns in response to salinity in the gills and kidney of milkfish may have a direct bearing on osmoregulatory functions of the two organs. Further studies are necessary to determine the role of specific lipids or fatty acids in osmoregulation of milkfish. In a similar study on guppy, Poecilia reticuiata, the total lipid, the ratios of phospholipids to neutral lipids and the fatty acid composition were affected by the rearing salinity (Daikoku et al., 1982). On sea water adaptation, there was an increase in total phospholipids with concomittant decrease in neutral lipid levels in the whole body. eye, gills, digestive tract, liveg and kidney as was observed in milkfish. Unlike in milkfish, where salinity did not affect total lipid content, total lipids increased in gills, digestive tract an guppy on seawater adaptation. Differences in fatty acid composition in response to salinity have been observed in migratory fishes. The percentage of (n-3) to (rr-6) fatty acids in sweet smelt (~~ecugfmsus dtivelis) decreased dramatically within 1 month after migration from the sea to freshwater (Ota and Tagaki, 1977 )- Opposite
88
LG. BORLONGAN AND L.V. BENITEZ
changes were observed in the salmon (Oncorhynchus ma& as they migrated from freshwater to seawater (Ota, 1976). The role of polyunsaturated fatty acids (PUFAs) in membrane permeability and plasticity may be one of the factors accounting for the differences in content of this family of fatty acids between freshwater and seawater-reared fishes. The highly unsaturated fatty acids in the phosphclipids found in organs of SW-reared milkfish could be a means of increasing membrane unsaturation and fluidity. Fluidity of membrane lipids is also related to the length and unsaturation of phospholipid fatty acyl chains (Kimelberg and Papahadjopoulus, 1972, 1974; Brenner, 1984; Sheridan, 1988). The n-3 fatty acid structure al!owa a greater degree of unsaturation than do the n-6 or n-9 family series. Information on lipid composition of fish can be used to make inferences about dietary requirements (Castell, 1979). An important implication of the effects of salinity on fatty acid composition is that milkfish reared in seawater are likely to have different essential fatty acid requirements than milkfish reared in freshwater. Seawater-reared milkfish may have a higher requirement for n-3 fatty acids than freshwater-reared milkfish. Thus, the culture of milkfish in different salinities may require feeds which differ in lipid and fatty acid composition. Further studies are necessary to determine the specific lipid and essential fatty acid requirements of milkfish both under SW and FW conditions. ACKNOWLEDGEMENT
The authors acknowledge with thanks the statistical and computer assistance of Ms. Larni Espada, Thanks also to Dr. R.M. Coloso for his suggestions and criticisms on the manuscript prior to submission.
REFERENCES Ackman, R.G., 1967. Characteristics of the fatty acid composition and biochemistry of some freshwater fish oils and lipids in comparison with marine oils lipids. Comp. Biochem. Physiol., 22: 907-922. American Public Health Association (APHA), 1976. Standard Methods for the Examination of Water and Wastewater. American Water Works Association, Water Pollution Control Federation, Washington, DC, USA. Bligh, E.G. and Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37: 91 I-9 17. Brenner, R.R., 1984. Effect of unsaturated fatty acids on membrane structure and enzyme &inztics. Prog. Lipid Res., 23: 69-96. Castell, J.D., 1979. Review of lipid requirements of fin&h. In: J.E. Halver and K. Tiews (Editors), Proc. World Symp. Finfish Nutrition and Fishfeed Technology, Hamburg, Germany, Vol. 1. Heenemann, Berlin, pp. 50-84.
LIPID AND FATTY ACID COMPOSITION OF MILKFISH
89
Cowey, C.B. and Sargent, J.R., 1977. Lipid nutrition in fish. Comp. Biochem. Physiol., 57B: 269-279. Crear, D., 1980. Observations on the reproductive state of milkfish population (Chanos chm) from hypersahne ponds on Christmas Island (Pacific Ocean ). Proc. World Maricult. SW., I I: 548-556. Daikoku, T., Yano, I. and Masui, M., 1982. Lipid and fatty acid composition and their changes in the different organs and tissues ofguppy, Poe&u reticulataon seawater adaptation. Comp. Biochem. Physiol., 73A: 167- 174. Dalagan, C.B., 1980. Fishpen operation in Laguna Lake. Greenfields, 10: 4- 11. ’ Dendrinos, P. and Thorpe, J.P., 1987. Experiments on the artificial regulation of amino acid and fatty acid contents of food organisms to meet the assessed nutritional requirements of larval, post-larval and juvenile Dover sole [ Solea soleu ( I_..) 1. Aquaculture, 6 1: 12 l-l 54. Fraser, A.J., Sargent, J.R., Gamble, J.C. and MacLachlan, P., 1987. Lipid class and fatty acid composition as indicators of the nutritional condition of larval Atlantic herring. Am. Fish. Sot. Symp., 2: 129- 143. Gruger, E.H., Nelson, R.W. and Standby, M.E., 1964. Fatty acid composition of oils from 21 species of marine, freshwater fish and shellfish. J. Am. Oil Chem, Sot,, 4 1: 662-667. Kimelberg, H.K. and Papahadjopoulos, D., 1972. Phospholipid requirements for (Na+ +K+ )ATPase activity: head-group specificity and fatty acid fluidity. Biochim. Biophys. Acta, 5 15: 209-237. Kimelberg, H.K. and Papahadjopoulos, D., 1974. Effects of phospholipid acyl chain fluidity, phase transition and cholesterol on (Na+ + Ki- ) stimulated adenosine triphosphatese. J. Biol. Chem., 249: 1071-1080. Metcalfe, L.D., Schmitz, A.A. and Pelka, J.R., 1966. The rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Anal. Chem., 38: 5 14. Moerck, E.K. and Ball, H.R., Jr., 1973. Lipids and fatty &ids of chicken bone marrow. J. Food Sci., 38: 978-980. Ostrowski, A.C. and Divakaran, S., 1989. The amino acid and fatty acid compositions of selected tissues of dolphin fish (Coryphaena hippurus) and their nutritional implications. Aquaculture, 80: 285-299. Gta, T., 1976. Lipids and masu salmon. IV. Changes of lipid composition and fatty acid composition in fles,h !ipids ofjuvenile masu salmon in the stages of seawater life. Bull. Fat. Fish. Hokkaido Univ., 27: 30-36. Ota, T. and Tagaki, T., 1977. A comparative study on the lipid class composi*ion and fatty acid composition of sweet smelt (Hecoglossusaltiwelis)from marine and freshwater habitat. Bull. Fat. Fish. Hokkaido Univ., 28: 47-56. Pullin, R.S.V., I98 1. Fishpens of Laguna de Bay, Philippines. ICLARM Newsletter, 4( 4 1: I I 13. Ringo, E. and Nilsen, B., 1987. Hatchery-reared landlocked Arctic charr, Salvelinus alpinus (L.), from Lake Takvatn, reared in fresh and sea water. I. Biochemical composition of food and lipid composition of fish reared in fresh water. Aquaculture, 67: 343-35 1. Sheridan, M.A., 1988. Lipid dynamics in fish: aspects of absorption, transport, deposition and mobilization. Comp. Biochem. Physiol., B90: 679-690. Snedecor, G.W. and Cochran, W.G., 1980. Statistical Methods, 7th edn. Iowa State University Press, Ames, IA, 507 pp.. Standsby, M.M.. 1967. Fatty acid patterns in marine, freshwater and anadromous fish. J. Am. Oil Chem. Sot., 44: 64-74. Stickney, R.R. and Hardy, R.W., Y989. Lipid requirements of some warmwater species. Aquaculture, 79: 145- 156. Teshima, S., Kanazawa, A., Horinouchi, K., Yamasaki, S. and Hirata, I-L, 1987. PhowhoWds of rotifers, prawn and larval fish. Nippon Suisan Gakkaishi, 53 (4 1: 609-6 15. Watanabe, T., 1982. Lipid nutrition in fish. Comp. Biochem. Physiol., B73: 3-l 7.
79
I.G. Borlongan and L.V. Benitez Aquaculture Department, SEAFDEC, Iloilo City, Philippines
(Accepted 5 September 1991)
APSTRACT
Borlongan, I.G. and Benitez, L.V., 1992. Lipid and fatty acid composition of milktish ( Chanos chanos Forsskal) grown in freshwater and seawater. Aquaculture, 104: 79-89. The lipid and fatty acid compositions of the various organs of milkfish fed with an invariant diet and reared in seawater (SW) and freshwater (FW) were determined using column chromatography and gas chromatography. Phospholipid content of the gills, kidney, liver, intestines and depot fat was higher in SW than in FW while the organs from fish in FW had higher contents of neutral lipid. Fatty acid patterns of total lipids in the liver, intestines and depot fat of mi!kf ;h reared in FW and SW were similar. There were marked differences in fatty acid patterns of gills.zikd kidn:y. The proportions of saturated to unsaturated fatty acids in gills and kidney were lower in SW than in FW. Likewise, the ratio of n-3 to n-6 fatty acids and total polyunsaturated fatty acids ( PUFAs) [Jfgills and kidney were higher in SW than in FW. The fatty acid patterus of the phospholipid fractionr, showed that SW-reared milkfish have higher total PUFAs, especially of the n-3 fatt;) acids, than the FW-reared milkfish not only in gills and kidney but in all organs examined. The differences in lipid ard fatty acid composition reflect a physiological response to the salinity in which milkfish were reared.
INTRODUCTION
The growing interest in farming milkfish both in brackishwater ponds and freshwater pens requires further insight into fish biochemistry and physiology during changes in salinity. The milkfish ( Charmschanos Forsskal ) is a euryMine species. It can thrive in natural waters of salinity ranging from 0 to 100 ppt (Crear, 1980; Dalagan, 1980; Pullin, 198 1). It is most comtionl~ cultured in brackishwater ponds where its ability to withstand wide and rapid changes in salinity is well-known. Lipids and fatty acids pray an important role in membrane biochemistry Correspondence to: LG. Borlongan, Aquaculture Department, Southeast Asian Fisheries velopment Center, P.O. Box 256, Iloilo City, Philippines.
0044~8486/92/$05.00
0 1992 Elsevier Science Publidrers B.V. All rights reserved.
De-
80
LG. BORLONGAN AND L.V. BENlTEi!
and therefore have direct impact on such membrane-mediated processes as osmoregulation, reproduction, nutrient assimilation and transport. Several studies have shown the usefulness of tissue fatty acid profiles in predicting the essential fatty acid needs of cultured fish (Castell, 1979 ). The fatty acid profiles of marine fish eggs (Dendrinos and Thorpe, 1987 ), larvae (Dendrinos and Thorpe, 1987; Fraser et al., 1987; Teshima et al., 1987 ), and juveniles (Ring0 and Nilsen, 1987; Ostrowski and Divakaran, 1989) have been used to predict the dietary requirements of fishes. Several studies have also shown that. the fatty acid composition of the diet influences that oi the fish (Cowey and Sargent, 1977; Watanabe, 1982; Stickney and Hardy, 1989). In additi,n to dietary considerations, the fatty acid composition of the fish is affected by environmental factors. The differences may be related to a specific requirement in fish enabling them to adapt physiologically to the environment. Although there are probably fish species with specific essential fatty acid (EFA) requirements, the influence of environmental factors is so great that the EFA requirements of a single species are changed by variation in temperature and salinity (CastelI, 1979). Studies have shown that salinity of water affects the fatty acid composition, especially the PUFA levels of fish. The ratio of n-3 to n-6 fatty acids is much lower in fish that thrive in FW than in a SW environment. These studies showed that the ratio of n-3 to n-6 fatty acids ranges from 1.7 to 3.5 and from 7.5 to 19.5 for freshwater and marine species respectively (Gruger et al., 1964; Ackman, 1967; Standsby, 1967). Whether these patterns are due to the effect of salinity alone or to differences in the natural foods present in these environments needs to be determined. The purpose of this work is to determine the lipid composition and fatty acid profile of the various organs of milkfish fed the same diet but cultured in freshwater and seawater. This information might prove both theoretically im-
portant to explain adaptation of euryhaline fish to salinity changes, and practically important as an aid to diet formulation in aquaculture. MATERIALS AND METHODS
Fish, salinityacdimation and experimentalrurt Milkfish weighing about 150-l 75 g were collected from fishponds with a salinity of 3 1 ppt and were transported to the laboratory. The fish were stocked in four canvas tanks filled with 10 tons of seawater. Each tank was stocked with 120 fishes. The fish were acclimated to laboratory conditions for 1 week and trained to accept pelleted feeds. At the end of this period, the salinity in two of the culture tanks was gradually reduced at the rate of 2 ppt per day by the introduction of freshwater until the salinity was about 0 ppt (FW ). The salinity of the two other tanks was maintained at full strength seawater (32 PP4 SW.
Both groups of fish were fed twice daily with the same commercial diet
LIPID AND FATTY ACID COMPOSTION
OF MILKFISH
81
(40% crude protein) at the raLe of 5% biomass per day. After 47 days from stocking, the t,wu groups of fish were fully acclimate to the salinity in their respective culture tanks and fully adapted to pelleted feeds. The fish were reared for another 60 days beyond full acclimation, for a total of 107 days. The water in both tanks was supplied by a flow-through system and supplemental aeration was provided. Selected physico-chemical parameters of the culture water, namely pH, dissolved oxygen, salinity, temperature, ammonia, nitrite and phosphate levels were determined thrice weekly using standard methods (APHA, 1976). Lipid analysis Samples of 30 fish each were collected at the start, at day 30, and at day 60 after full acclimation from- each tank of fish reared in freshwater (FW) and seawater (SW ) . Individual weight of fish was recorded and the fish were then dissected to isolate five different organs, namely. gills, kidney, liver, intestines and depot fat. Identical organs from each batch of 30 fish per tank were pooled and total lipid content of each organ was determined by the method ( 1959 ). Duplicate analyses were carried out on each pool. The total lipid was further resolved into neutral lipid, phospholipid and glycolipid fractions by silicic acid column chromatography using the method at 0.05% was of Moerck and Ball ( 1973 ) . utylated hydroxytolu&e ( tal lipid fracadded to all solvent systems prevent lipid oxidation. tion was converted into fatty acid methyl esters by saponification with 0.5 N and transesterified with boron trifluoride methanol remethanolic Na agent (Metcal al., 1966 ). The fatty acid methyl esters were analyzed on a Shimadzu CC-4C gas chromatograph equipped with a flame ionization detector and using a stainless steel 15% diethyleneglycol succinate mosorb W support. he column temperature was set at 180°C and fatty acids were identified by comparison with known standards. Ch dards of polyunsa.turated fatty acid methyl esters (No. 7015) were purchased from Supelco, USA. All other reagents we ical grade. Statistical analysis One-way analysis of variance and uncan’s multiple range test were use for statistical comparison of results between organs and Student’s t-test for the comparison of results between treatments (freshwater versus seawater) (Snedecor and Cochran, 1980). Differences were considered significant at Pc 0.05 level. RESULTS
Physics-chemical parame,“ers The analyzed physico-chemical parameters of the culture water were within tolerable limits. Dissolved oxygen ranged from 4.4 to 7.4 ppm in the seawater
LG. BORLONGAN AND L.V. BENITEZ
82
tank and 5.5 to 9.1 ppm in the freshwater tank. The concentrations of nitrite and ammonia ranged from zero to negligible values for both fresh and seawater tanks for the duration of the feeding trial. Temperature ranged from 26 to 32 “C and pH from 7.6 to 8.4 for both fresh and seawater ta.nks. The seawater tank had a salinity of about 27 to 32 ppt and the freshwater ‘rtinkhad zero or negligible salinity. Effect of salinity on lipid composition
Total lipid content expressed as percentage of wet tissue weight of the various organs of milkfish reared in freshwater and seawater is shown in Table 1. In the initial pond samples, the highest lipid content was observed in the gills. After the 30-day culture period, the lipid content of the gills and kidney in both FW and SW reared fish were comparable with the initial samples. However, there was a significant increase in the total lipid content in the depot fat and liver but a significant decrease in the intestines. At the end of the 60-day culture period, significant increase in the total lipid content was observed in the depot fat, liver and gills but there was a significant decrease in the kidney and intestines for both the FW and SW reared fish. At both day 30 and 60, there was no significant difference in the total lipid content in all organs between FW and SW reared fishes. Results of the fractionation (Tables 2-4) of the total lipids showed that of the three lipid classes, the neutral lipid (65-78%) was predominant in all organs of ftsh reared in both freshwater and seawater. This was followed by phospholipids ( 13-26%) and glycolipids ( 5- 11%). No significant differences were found in the % neutral lipid content of the depot fat and liver between seawater and freshwater reared fish (Table 2). However, the gills, kidney and intestines from fish reared in freshwater had significantly higher TABLE I Total lipid content of various organs of milkflsh reared in freshwater and seawater Organs
Total lipids’*2 (g/ 100 g wet weight tissue) Initial pond samples
Day 30 FW
Gills Kidney Liver Intestines Depot fat
7.5 kO.9’ 4.3 + 0.3” 4.3%0.5” 3.6+0.7a 5.7 -i-0.6b
8.6f0.3’ 5.3f0Sb 6.0 + 0.3b l.4+0.8a Il.O& 1.5d
Day 60 SW
FW
SW
8.7f0.4C 9.6&0.3b 9.7+0.5b 4.7 f 0.4b 1.1kO.3” 1.9* 1.0” 5.7+0.5b 10.9f 1.3b 13.0 + 2.4’ 2.2 + o.7a 1.1f0.5” I .4 + 0.8” 9.82 1.5’ 16.01t 1.3’ 14.6+ 1.6d .-_ ‘Means not sharing a common superscript in each column are significantly different at P< 0.05. “No significant differences in all organs between FW aud SW reared milk&h at PC 0.05.
LIPID AND FATTY ACID COMPOSITION OF MILKFISH
83
Neutral lipid content of various organs of milktish reared in freshwater and seawater Organs
Neutral lipids’ (% of total lipids) Initial pond samples
Gills Kidney Liver Intestines Depot fat
66.5 * 0.3” 66.7kO.2” 66.4 + 0.4a 67.7 f 0.5b 72.5 k 0.6’
Day 30
77.9+0.3e** 75 I +04d** 73:4 7 1:570.6 ? 0.3a++ 72.8 + 0.3b*“s
Day 60 SW
FW
SW
66.1 I!I 0.3b 68.1+0.7’ 70.9 f 0.7” 65.0&0.8” 72.1 kO.9’
75.2 +0.3d** 72.3 f 0.4’~” 7 1.6 + 0.5b*“S 69.3 f 0.7”*” 72.3 + 0.9’.“”
65.9 + 0.6b 64.7 +0.8” 71.2f0.5d 66.5 I!I0.9’ 71.6f0.7’
‘Means not sharing a common superscript in each column are significantly different at P-c 0.05. *Highly significant; “’ no significant differences between FW axi SW reared fish at P~0.05. TABLE 3 Phospholipid content of various organs of milkfish reared in freshwater and seawater Organs
Phospholipids’ ( W of total lipids) Initial pond samples
Gills Kidney Liver intestines Depot fat
23.1 t-0.1’ 22.6 ?I0.3” 25.9 + 0.4* 21.7tO.l” 22.4kO.l”
Day 30
Day 60
FW
SW
FW
SW
13.2-t 0.4’*** 14.0+ 0.4b*L* 18.1!I 0.4’** 18.4f 0.5c**+ 20.4 It 0.3d**
24.910.5’ 22.2+0.5b 20.9 + 0.3’ 25.7 etlO.Sd 22.5 kO.5”
16.3 + 0.48*** 18.1? 0.4b*+* 19.7 -t 0.4’,* 20.0 I?I0.2’,* 20.8 Z!I OS”*’
25.1 kO.3’ 25.8+0.5’ 20.7 III0.5” 23.0+0.5” ZE.7 II 0.4”
‘Means not sharing a common superscript in each column are significantly different at P-z 0.05. **Highly significant; + significant differences between FW and SW reared milkfish at PC 0.05.
percentage contents of neutral lipids than those of fish reared in seawater. The gills appeared to have the highest content of neutral lipids for the freshwater samples while the depot fat and liver had the highest level of neutral li the seawater samples. Milkfish reared in seawater had a significantly higher content of phosp lipids compared to those reared in fres initial fish samples obtained from the fi to that of the seaw to the samples rear a level of 13 to 2 1% for 21 to 26% in organs o
84 TABLE
LG. BCRLONGAN
AND L.V. BENITEZ
4
Glycoiipid content of various organs of milkfish reared in freshwater and seawater Organs
Glycolipids’*’ (96of total lipids) Initial pond sampies
Gills Kidney Liver Inteaines Depot fat
10.4 +_0.4’ 10.7fO.l’ 7.7+0.1b 10.6+0.7’ 5. I kO.4”
Day 60
Day 30 FW
SW
FW
SW
8.9+_0.5b io.9+ 1.F 8.5 fOSb I l.Of 1.7’ 6.9+ 1.3;’
9.010.5’ 9.7 + I .O’ 8.2 + 0.4b 9.3 +osc 5,4+ 1.5”
8.4 + 0.6b 9.6 + 0.5b 8.8 + 0.6b 10.7+0.8’ 6.9?0.5”
8.9 + 0.4b 9.5&0Sb 8.1 kO.7” 10.5?.0.4’ 5.7 -t 1.3”
‘Means not sharing a common superscript in each column are significantly different at P-z 0.05. ‘No significant differences in all organs between FW and SW reared fish at PC 0.05.
those reared in freshwater. The gills had lowest levels of phospholipid for fish reared in freshwater. Comparison of the glycolipid content of the various organs of milkfish reared in freshwater and seawater showed no significant differences (Table 4). In both groups of fish, significantly higher glycolipid content was observed in the intestines, kidney and gills (the organs associated with high numbers of epithelial cells), while the depot fat had the lowest glycolipid content.
omparison of fatty acid patterns of the total lipids from various organs of fishes reared in freshwater and seawater showed similar patterns in the liver, intestines and depot fat for both the Day 30 and Day 60 samples. However, marked differences were noted in the fatty acid patterns of the gills and kidneys, particularly for the Day 60 samples (Table 5 ). The gills from seawaterreared fishes contained large quantities of unsaturated fatty acids (72.5%) and much lower quantities of saturated fatty acids (27.5%). The opposite was observed in the freshwater samples where the gills contained larger quantities of saturated fatty acids (84.9%) than unsatu fatty acids ( 15.0%). The gills of SW-reared fish contained higher pert es of 16: 1 and 18: 1 FAs than the R&reared fish. In the kidney, the predominance of saturated fatty acids over unsaturated fatty acids in the freshwater samples was noted. Unlike the hills: this same trend was observed for the seawater samples. In both gills and kidney, the ratio of (n-3) to (n-6) fatty acids and the polyunsaturated fatty acid (PUFA) content were h in the seawater than in freshwater-reared fish. For both the FW and S red fish the predominant saturated FA in all organs was stearic ( 18 : 0) acid.
__
7.98* 1.49 5.36* 76.15 23.84* 9.47* 3.19
3.98 2.35 1.69 85.69* 14.29 6.33 5.99* 6.50 2.37 2.74 76.46 23.54 8.87 3.25
5.90 7.49 4.46 13.14 58.61* 1.02 1.11* 2.08 0.50 0.41 0.84 4.30 0.01 0.03 0.09
Intestines
6.40 1.67 3.83 78.88 21.12 8.07 3.73
11.86 8.50 3.48 10.98 55.04 0.66 0.42 1.80 1.34 0.42 0.83 4.51 0.07 0.06 0.03
SW
2.80 1.42 1.97 70.33 29.66 4.22 2.37
5.32 5.78 10.27* 10.72 48.96 14.42* 0.39 0.63 0.23 0.18 0.85 1.88 0.07 0.09 0.20
FW
Depot fat
2.07 1.45 1.43 78.66 21.35 3.52 3.68
5.81 lO.28* 2.92 16.57* 59.65 1.02 0.50 0.55 0.22 0.20 0.75 1.48 0.02 0.02 0.02
SW
4.05 1.66 2.44 80.82 19.16 5.71 4.22
1.60 4.06 10.34 12.57* 64.82 0.48 1.09 0.55 0.09 0.25 0.32 3.00 0.31* 0.07 0.43
Liver
7.05* 2.10 3.36 83.36 16.63 9.15’ 5.01
4.61 8.20’ 10.02 7.18 60.53 0.14 1.45 0.71 0.09 0.22 0.43 6.28* 0.07 0.03 0.03
SW
-
_.-_
-”
-
I
_
-
.
. -
‘Day 60 samples. *Composition expressed as weight % of total lipids. Values are me&s of four replicate znalyw-YS:r!ndard deviations are omitted for clarity ofpresentation but normally were c 5%. *Highly significant differences between FW and SW reared milkf:sh at PC 0.05 (Student’s r-test ).
Total satd FAs Total unsatd FAs Total PUFAs Satd/unsatd
(n-3) (n-9) (n-6) (n-6) (n-3) (n-9) (n-3) (n-3)
1.64 1.00 1.64 84.98+ 15.01 2.64 5.66
18:3 2O:l 20:3 20:4 20:s 22:1 22: 5 22:6
Total (n-3) I-As Total (n-6 ) FAs n-3/n-6 FAs
4.46 15.79 19.87* 12.72* 36.03 0.49 0.40 3.34* 1.09* 0.33 0.79 4.56* 0.07 0.04 0.04
3.24 14.44 3.90 6.76 64.11* 0.86 1.70* 1.37 0.17 0.24 0.41 2.43 0.17 0.03 0.15
0.14 2.06 0.71 42.59* 24.57 20.92* 1.16* 0.48 0.56 0.17 0.67 0.23 0.33* 1.88* 3.51* 6.10* 2.00 3.05* 27.48 72.50* 8.10* 0.36
2.00 14.84” 5.88* 11.85 62.26* 0.37 0.38 0.68 0.13 0.12 0.50 0.85 0.02 0.04 0.07
FW
SW
FW
SW
Kidney
Gills
Percentage composition*
12:O 14:o 16:O 16:l (n-7) 18:O 18:l (n-9) 18:2 (n-6)
Fatty acids
Fatty acid composition of the total lipids from various organs of milkfish reared in freshwater and seawater’
TABLE 5
0.79
21.29 23.57 10.83 30.43* 0.49 4.06* 0.49 0.32 0.4 1 0.99 0.35 3.22* 1.39* 9.66* 1.22 7.92* 34.28 6j.?2* 10.88*
0.52
0.88
1.48
0.23 1.84 20.86 21.30 21.11* 24.25* 0.43 1.53 1.48 0.78 0.65 0.30 1.98. 2.46 0.80 5.09 1.86 2.74 44.04 55.96 6.95
1.01 5.22 25.94 25.19 14.50 20.98 1.05 1.39 0.26 0.29 0.19 1.07 0.87 1.89 0.15 4.50 1.53 2.94 46.67* 53.33 6.03
0.68
FW
FW
SW
Kidney
Gills
Percentage composition*
0.83
0.63 6.44” 24.79 24.22 13.51 14.87 1.11 1.51 3.54* 0.82 0.22 0.51 1.51 3.92* 2.40* 8.34* 2.15 3.88” 45.37 54.63 10.49*
SW
13.26* 1.97 6.73* 41.37 58.63* 15.23* 0.71
7.06 2.14 3.30 50.78* 49.22 9.20 1.03
0.83 0.65 2.97 1.78
1.27*
0.79 0.85 30.66 25.02* 9.07 17.24 0.91 2.51* 0.37 0.52 0.54 5.38* 0.77 2.54 3.19*
SW
0.54 1.29 40.72* 17.62 8.23 17.80 0.72 1.48 3.95* 0.15
FW
Intestines
SW 0.18 0.12 4:.11* 19.03 3.97 12.77 0.62 1.06 5.55* 0.93 0.25 1.13* 0.67 ?.23* 5.38* 14.80* 1.80 8.22* 45.38 54.62 16.60* 0.83
FW 0.06 0.16 29.94 34.98* 10.73* 17.15 0.85 1.60 0.19 0.20 0.13 0.22 0.29 3.20 0 30 5.32 1.18 4.51 40.89 59.11 6.50 0.60
0.85 3.01 11.71 26.39 23.68* 26.14* 1.os* 2.54* 1.18* 0.42 0.78 2.61 0.03 0.05 0.07 5.27* 2.28* 2.31 38.7 1 61.29* 7.55* 0.63
0.91 0.48 35.42* 29.26 10.95 17.74 0.34 1.32 0.36 0.29 0.72 2.07 0.02 0.04 0.08 3.51 1.35 2.60 47.76* 52.24 4.86 0.9 1
Liver SW
FW
Depot fat ---
. . .. .
. . .. .
. __-
_ ._
_ ,_
_
as
,
_,
I
--
_
.._
‘Day 60 samples. ‘Composition expressed as weight Z of total phospholipids. Values are means of four replicate analyses. Standard deviations are omitted for clarity of presentation but normally were < 5%. *Highly significant differences between FW and SW reared milktish PC 0.05 (Student’s t-test ).
Satd/unsatd
12:0 14:o 16:0 16:1 (n-7) 18:O 18:l (n-91 t8:2 (n-6) 18:3 (n-3) 2O:l (n-9) 20:3 (n-6) 20:4 (n-6) 20: 5 (n-3) 22: 1 (n-9) 22:5 (n-3) 22:6 (n-3) Total (n-3) FAs Total (n-6) FAs n-3/n-6 FAs Total satd FAs Total tmsatd FAs Total PLJFAs
Fatry acids
Fatty acid composition of the phospholipids from various organs of milkfish reared in freshwater arnd seawater’
TABLE 6
8 g z $ g r .< g z =i A
5 $ F
_
_.
LlPlD AND FATTY ACID COMPOSLTION OF MILKFISH
87
Effect of stalinity on the fatty acid composition of phosphdipid frmions While salinity affected only the fatty acid patterns of the total lipids gills and kidney, it affected the fatty acid patterns of “ihephospholipid fractions of all organs examined (Table 6). There was a significantly higher centage of total PUPAS in all organs for the SW-reared fish compared to reared fish. The phospholipids from organs of SW-reared fish contained h percentages of n-3 fatty acids than FW-reared fish. Consequently, the ratios of n-3 to n-6 fatty acids of phospholipids from SW-reared fish were also higher. DlSCIJSSION
NO significant differences in the percentage total lipid content were observed in all organs of milkfish reared in FW and SW, indicating that salinity did not affect fat deposition in terms of amount of total lipid stored. HOWever, the composition of the different lipid classes ( phospholipid, neutral lipid and glycolipid) and fatty acids of milkfish changed with the salinity of the rearing water. The differences in the lipid and fatty acid composition in response to salinity were expressed principally in the gills, ki neys and intestines of milkfish. Phospholipid levels of gills, kidneys and intestines were 1of neutral lipids were higher in fishes reared in SW than in FW. Nigher 1 noted in those organs offish reared in FW than in Salinity significantly affected the fatty acid composition of the phospholipid fractions of all organs, while in the total lipids differences in fatty acid patterns were most pronounced in the gills and kidneys. The levels of unsaturated I%, ratio of (n-3) to (n-6) PAS and levels of PUFAs were higher in than in FW-reared fish. The change in lipid composition and fatty acid erns in response to salinity in the gills and kidney of milkfish may have a direct bearing on osmoregulatory functions of the two organs. Further studies are necessary to determine the role of specific lipids or fatty acids in osmoregulation of milkfish. In a similar study on guppy, Poecilia reticuiata, the total lipid, the ratios of phospholipids to neutral lipids and the fatty acid composition were affected by the rearing salinity (Daikoku et al., 1982). On sea water adaptation, there was an increase in total phospholipids with concomittant decrease in neutral lipid levels in the whole body. eye, gills, digestive tract, liveg and kidney as was observed in milkfish. Unlike in milkfish, where salinity did not affect total lipid content, total lipids increased in gills, digestive tract an guppy on seawater adaptation. Differences in fatty acid composition in response to salinity have been observed in migratory fishes. The percentage of (n-3) to (rr-6) fatty acids in sweet smelt (~~ecugfmsus dtivelis) decreased dramatically within 1 month after migration from the sea to freshwater (Ota and Tagaki, 1977 )- Opposite
88
LG. BORLONGAN AND L.V. BENITEZ
changes were observed in the salmon (Oncorhynchus ma& as they migrated from freshwater to seawater (Ota, 1976). The role of polyunsaturated fatty acids (PUFAs) in membrane permeability and plasticity may be one of the factors accounting for the differences in content of this family of fatty acids between freshwater and seawater-reared fishes. The highly unsaturated fatty acids in the phosphclipids found in organs of SW-reared milkfish could be a means of increasing membrane unsaturation and fluidity. Fluidity of membrane lipids is also related to the length and unsaturation of phospholipid fatty acyl chains (Kimelberg and Papahadjopoulus, 1972, 1974; Brenner, 1984; Sheridan, 1988). The n-3 fatty acid structure al!owa a greater degree of unsaturation than do the n-6 or n-9 family series. Information on lipid composition of fish can be used to make inferences about dietary requirements (Castell, 1979). An important implication of the effects of salinity on fatty acid composition is that milkfish reared in seawater are likely to have different essential fatty acid requirements than milkfish reared in freshwater. Seawater-reared milkfish may have a higher requirement for n-3 fatty acids than freshwater-reared milkfish. Thus, the culture of milkfish in different salinities may require feeds which differ in lipid and fatty acid composition. Further studies are necessary to determine the specific lipid and essential fatty acid requirements of milkfish both under SW and FW conditions. ACKNOWLEDGEMENT
The authors acknowledge with thanks the statistical and computer assistance of Ms. Larni Espada, Thanks also to Dr. R.M. Coloso for his suggestions and criticisms on the manuscript prior to submission.
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