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Aquatic Ecology 37: 169–182, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.
Effects of algal diets and temperature on the growth and fatty acid content of the cichlid fish Oreochromis niloticus L. – A laboratory study Zenebe Tadesse 1, Merike Boberg 2, Lars Sonesten 3 and Gunnel Ahlgren 4,* 1
National Fisheries and other Living Aquatic Resources Research Center, P.O. Box 64, Sebeta, Ethiopia; Department of Public Health and Caring Sciences/Geriatrics, Uppsala University, Box 609, Uppsala, SE-751 25, Sweden; 3Department of Environmental Assessment, Swedish University of Agricultural Sciences, P.O. Box 7050, Uppsala, SE-750 07, Sweden; 4Department of Limnology, Uppsala University, Norbyvägen 20, Uppsala, SE-752 36, Sweden; *Author for correspondence (e-mail:
[email protected]) 2
Received 7 February 2002; accepted in revised form 21 January 2003
Key words: Condition factor, Feeding experiments, Food quality, Nile Tilapia, Tropical fish Abstract The effects of different algal foods and water temperatures on the growth and fatty acid content of the Nile Tilapia, Oreochromis niloticus L., were studied. Four types of algae, given in the same amounts as the control diet, were used as food: Microcystis aeruginosa, colonial and single-celled forms; Arthrospira fusiformis; and Scenedesmus quadricauda. The control group was fed a commercial diet of cichlid pellets, while another group was left unfed. The feeding experiment was run at 25 °C. The condition factor decreased in all algal fed fish groups, except the one fed on Microcystis colonies, whereas the control group showed no significant change. Both food quantity and quality were responsible for this result. Some short-chained fatty acids in the diets could be traced in the long-chained counter-parts in the fish tissue. Both saturated fatty acids and monounsaturated fatty acids were higher in the control vs. treatment groups, whereas the polyunsaturated fatty acids displayed no significant differences amongst any of the treatment groups studied, including the unfed group. Direct quantitative comparison of individual fatty acid in the diet vs. tissue lipids in the fish proved to be difficult due to the great capacity of these tilapias to elongate and desaturate 18 carbon acids into long-chained homologues. The effect of temperature was studied by growing the fish at 16, 20 and 25 °C. All groups were fed commercial cichlid pellets. The level of saturated and monounsaturated fatty acids increased at 20 °C, whereas polyunsaturated fatty acids showed little variation. Docosahexaenoic acid, belonging to the important ‘omega 3’ group where the first double bond starts at carbon number three, was highest at 16 °C, resulting in a markedly elevated omega-3/omega-6 ratio at that temperature. Introduction It is now well established that the lipid and fatty acid (FA) contents of fish vary a great deal both within and between species (El-Sayed et al. 1984; Ahlgren et al. 1994, 1999; Zenebe et al. 1998a, 1998b). Various factors such as diet, temperature, genetic variation, age, size and season have been suggested to be responsible for the observed variability (Henderson and Tocher 1987). In our initial study, we found a 10-fold variation in the FA and lipid contents of Oreochromis niloticus L. collected from various lakes in Ethiopia
(Zenebe et al. 1998a). We suggested diet composition to be the main factor regulating the observed variations. Both food quality and quantity are known to influence the composition of fatty acids in fish. Starvation is also known to affect the fatty acid composition of body tissues, particularly in the liver, muscle and visceral fat deposits (De Silva et al. 1997). However, the nature of the response to food deprivation differs from species to species with regards to the type of reserves utilised and the tissue from which they are drawn (Steffens 1989).
170 Oreochromis niloticus is reportedly capable of elongating and desaturating the 18 carbon polyunsaturated fatty acid (PUFA), i.e., linoleic acid (LA, 18:26) and ␣-linolenic acid (ALA, 18:33), into their long-chained homologues, the biologically active forms arachidonic acid (ARA, 20:46), eicosapentaenoic acid (EPA, 20:53) and docosahexaenoic acid (DHA, 22:63) (Olsen et al. 1990; Sargent et al. 1995). However, in the presence of performed 20 and 22 carbon PUFA in the diet, the rates of conversion of 18 carbon PUFA to long-chained derivatives would be expected to be less than maximal and an accurate pattern of substrate preference for certain FA may not be observed (Olsen et al. 1990). In addition to diet, temperature has also been reported to influence the FA composition of planctonic crustacea and fish by either inducing or deactivating desaturases (Farkas et al. 1981; Farkas 1984). Farkas suggested that lower temperatures may induce the formation of long-chained FA in fish. This explanation was based on the homeoviscous adaptation of membrane fluidity to temperature change (e.g., Hazel (1984)). Hazel (1984) also suggested that this response is so quick that current temperature is more critical to the production of PUFA than is the life history of the fish. However, we hypothesized that the effects of temperature may be negligible in thermophilic fish adapted to the high water temperatures of the tropics. Diet may be a far more critical factor in determining the fatty acid contents of these fish. Thus, in the present study the authors assess the effect of four different mono-algal diets and three temperatures on the growth and FA contents of Oreochromis niloticus grown under controlled conditions in the laboratory. The following questions were addressed: 1. Can fatty acids (FA) of mono-algal diets be traced in the FA patterns of Oreochromis niloticus? 2. Is diet composition a regulating factor of growth and FA quality in Oreochromis niloticus? 3. Does temperature influence the FA content of Oreochromis niloticus?
Materials and methods Experimental set-up Oreochromis niloticus-fry were obtained from the University of Stirling, Aquaculture Institute in Scotland. The fry were maintained in the laboratory in 40
L aquaria for four months at 25 °C and fed commercial feed (Nippon Cichlid Basic Pellet), until the experiment was started. For each treatment, 5–8 fish of approximately the same size were kept in 10 L aquaria. Despite some variation in the size of the fish, the mean length and weight did not differ significantly among the six fish groups (Figure 1, Initial). Before the start of the temperature experiment, the fish were acclimated to their respective temperatures for one week. In the diet experiments, four types of algae were used: Microcystis aeruginosa Kütz. (colonial), Microcystis aeruginosa Kütz. (single-celled), Scenedesmus quadricauda Turp (1–4 celled), Arthrospira fusiformis (Voronich) Komarek and Lund (1990) (= Spirulina platensis (Gom.) Gietl. (helicidal threads). All four species are common food for natural Oreochromis in Ethiopian lakes (Zenebe et al. 1998a). Fresh Microcystis colonies (MICR1) were collected in a bay of Lake Mälaren (Uppsala, Sweden) 2–3 times a week (temp. 14–21 °C). Test of algal toxicity in this bay gave values up to 30 µg microcystin/g dry weight (ELISA-test, Chorus and Bartram (1999)). The cultured form of Microcystis (unfortunately single-celled) was also used because the Microcystis bloom in the bay was not expected to last through the whole experiment. The single-celled algae Microcystis (MICR2, CCAP 1450/1, Windermere Lab., Ambleside, England, not marked as toxic) and Scenedesmus (SCEN) were grown in Z8⬘ medium (Staub 1961; Ahlgren 1977) in turbidostats at 25 °C. Arthrospira (SPIR) was also grown in turbidostats at 25 °C, but in Zarrouk’s medium, modified by George (1976) (see Kebede and Ahlgren (1996)). It was difficult to obtain enough algal material from the turbidostats alone, therefore, additional batch cultures were set up in which MICR2 and SCEN were grown in Z8⬘ medium for about one week before harvesting. The control group (CONT) was fed cichlid pellets (the same batch during the whole experiment). Another group was left unfed (STARV). The diet groups, including the unfed fish, were all kept at 25 °C. The temperature groups were all fed cichlid pellets and kept at three temperature regimes: 16 °C (15.7 ± 0.8), 20 °C (19.8 ± 0.5) and 25 °C (25 ± 0.5). The lowest temperature was chosen based on findings that Tilapia stops feeding below 16 °C (Chervinski 1982). The other two groups were run in constant-temperature rooms. The temperature groups were run at the same time as the diet groups, so the control (CONT) was the same as in the temperature group at 25 °C. The fish were fed during 8 weeks except for the MICR1 group, for which the
171 experiment lasted just three weeks, because the Microcystis bloom ended in the bay. The amount of food required to reach satiation, approximately 2% of their body weight, was determined with pellets and the same amounts (in carbon) of the algal feeds were used. The amount of algal food was judged to be sufficient, based on other feeding regimes of Tilapias (Balarin and Hatton 1979; McDonald 1985; Northcott et al. 1991). The food was provided twice a day to avoid sedimentation. Oreochromis is also a filter feeder, eating several times per day in nature (Tudorancea et al. 1988). The experimental period of 8 weeks was considered to be sufficient for young cichlids. In a parallel experiment, opaque macrozones in the otoliths, which form during active growth, were found in 60% of the fish after one month at 25 °C and in 100% of the fish after four months (Admassu 1998). Both the initial and final weights of each fish were recorded. As it was impossible to follow the growth of individual fish, the average initial and final fish length (L) and weight (W) were calculated. The condition factor, K = W × 100/L 3 (Weatherley and Gill 1987), was calculated using both the lowest and highest 95% confidence limits for L and W (average L and W ± t 0.025(n − 1) × s/冪n), which produced a confidence interval for K. The assumption was made that the maximum/minimum fish lengths corresponded to the maximum/minimum fish weights. No compensation for water content was made. Analyses of water content in 14 fish species from different lakes showed very constant dry weight/fresh weight ratios (0.20, CV = 0.01, n = 35) when the fatty eels were excluded (Ahlgren et al. 1994). At the end of the experiment, the fish were sacrificed and the dorsal muscle removed for fatty acid analysis. Three samples were randomly chosen from each group. The basic reason for performing fish studies in poorer countries like Ethiopia is ultimately related to the question of food resources. With that in mind and working on a limited budget, only the dorsal muscle was chosen for analysis over other more physiologically interesting tissues, e.g., liver and heart muscle. Fatty acid analysis The dorsal muscle tissue samples were freeze-dried and stored at −20 °C under nitrogen gas during a maximum of four months. Freeze-drying has been shown to be a very mild process that does not change the FA composition nor contents (Ahlgren et al. 1992, 1994). Possible negative effects of sample storage at
Figure 1. Change in total length (a), total weight (b), and condition factor (c) of Oreochromis niloticus fed cichlid pellets (CONT) and different algal diets (MICR1 = Microcystis colonies, MICR2 = single-celled Microcystis, SPIR = Arthrospira, SCEN = Scenedesmus quadricauda, STARV = unfed). Bars indicate standard deviation (SD) of the mean. No bars indicate that SD is too small to be shown.
−20 °C on FA concentrations were < 2% for storage up to 2 years (M. Boberg, unpublished data). Immediately preceding the FA analysis, the fish tissue was homogenised using a spice mill (Bamix, Casa, Sweden). Dried samples of the four algal food types, as well as the pellets, were also analysed for FA contents. FA were measured as their methyl esters using
172 gas chromatography (GC, QUADRIX with a 25 m silica capillary column number 80730F, ID = 0.32 mm, film thickness 0.24 µm) according to the procedure described in detail in Boberg et al. (1985) and Ahlgren et al. (1994). This column clearly separates nervonic acid (24:19) from DHA. Extraction of the pre-weighed samples was performed by homogenising them in methanol-chloroform, containing butylated hydoxytoluene (BHT) as the oxidant and NaH 2PO 4 as the buffer. The internal standard was added and the extract was kept at 4 °C for 1–2 days. The chloroform phase was pipetted off and the sample was evaporated to dryness under a stream of nitrogen. The lipid esters were transmethylated at 60 °C in 5% H 2SO 4 over night. The methyl esters were extracted in petroleum ether, containing some water and BHT; the solvent was evaporated again and re-dissolved in 1 ml Uvasol, grade hexane. The injection temperature was 230 °C and the temperature gradient 100–215 °C. The carrier gas was helium. Individual FA were identified by comparing their retention times with those of several commercially available standard mixtures: GCL-68A (Nu-Chek Prep, Inc.) mixed with Fish oil 30 (batch Q 80804 with 23 acids, Larodan Fine Chemicals AB). The FA were quantified by injecting fixed volumes of the dissolved, pre-weighed samples into the GC and comparing the area of the peaks with the peak of an internal standard recommended for fish samples, i.e., 0.25 or 0.10 µg per sample of tricosanoic acid (23:0) (Einig and Ackman 1987). Because FA analyses are costly, only single samples of the feed algae were analysed for this experiment. However, earlier duplicate analyses showed only small variations (0–10%) in individual FA with abundance ⭓ 10% of 兺FA, except for some green algae where variation was occasionally as much as 20–30% (cf. Ahlgren et al. (1992)). The results are expressed in mg g −1 dry weight (dw). No corrections were made to take into account that various FA may have different detector responses, depending on the chain length and number of double bonds. The internal standard was, however, chosen to give the same response as the PUFA, the most interesting FA in this study. Total FA in Tables 1, 2 and 3 were also calculated from the total areas integrated from all FA in the chromatograms. The difference between total FA and 兺FA, which could be measurements of unidentified acids/ substances, was not large in the fish samples, ⭐ 7% (Tables 2 and 3), but larger in the algal samples (16– 30%).
Table 1. Fatty acid content (mg g −1 dw) of commercial diet (pellets) and algae used in the study. MICR1 = Microcystis aeruginosa colonies, MICR2 = single-celled Microcystis, SPIR = Arthrospira fusiformis, SCEN = Scenedesmus quadricauda. Total FA are calculated from the total integrated areas of all FA in the chromatograms. Fatty acids
Pellets
MICR1(col) MICR2 SPIR
12:0 14:0 14:15 15:0 16:0I 16:0 16:17 17:0I 17:0 17:17 18:0I 18:0 18:19 18:17 18:26 (LA) 18:36 (GLA) 18:33 (ALA) 18:43 20:0 20:19 20:26 20:36 20:46 (ARA) 20:53 (EPA) 22:0 22:111 22:46 22:56 22:53 22:63 (DHA) 24:19
0.027 0.497 – 0.077 – 2.673 0.772 – 0.060 0.079 0.123 0.701 2.248 0.322 3.085 0.070 0.452 – 0.055 0.202 – – 0.083 0.572 – 0.275 – – – – 0.881
0.153 0.100 0.046 0.014 0.151 8.107 0.197 0.053 0.039 0.068 0.166 0.371 0.236 0.202 1.159 3.172 1.361 – 0.023 – 0.038 0.077 – – – – – – – – –
兺SAFA 兺MUFA 兺PUFA
4.21 3.90 5.23
9.13 0.75 5.93
10.69 1.30 10.64
9.24 1.68 9.49
17.11 6.61 29.58
13.34 1.99 3.24 0.61 0.39 14.36
15.81 1.48 4.45 0.33 0.38 22.77
22.63 0.95 9.72 0.10 0.47 26.31
20.41 0.05 9.23 0.01 0.47 25.89
53.30 19.99 9.58 2.1 0.56 63.31
兺FA 兺 3 兺 6 兺3/兺6 兺PUFA/兺FA Total FA
– indicates not found or < 0.02 mg g −1.
0.132 0.091 0.064 0.012 0.232 9.617 0.376 – 0.044 – – 0.544 0.135 – 1.844 7.407 0.361 0.480 – – 0.086 0.383 – – – – – – – – –
SCEN
– – 0.582 0.022 0.104 0.244 – 0.032 0.378 0.647 7.378 4.562 1.28 0.472 0.029 2.707 0.026 0.042 – – 0.509 8.748 0.339 0.104 0.230 3.747 0.068 0.602 1.603 8.901 7.504 0.607 0.053 18.45 – 1.154 – – – 0.059 0.126 – – – – – – – – – – – – – – – – – – – – –
173 Table 2. Fatty acid content, mean (n = 3) and standard deviation (mg g −1 dw), of Oreochromis niloticus dorsal muscle tissue supplied with different algal feed at 25 °C. Total FA are calculated from the total integrated areas of all FA in the chromatograms. The control, see Table 3, 25 °C. Fatty acid
MICR1
MICR 2
SPIR
12:0 14:0 14:15 15:0 16:0I 16:0 16:17 17:0I 17:0 17:17 18:0I 18:0 18:19 18:17 18:26 (LA) 18:36 (GLA) 18:33 (ALA) 18:43 20:0 20:19 20:26 20:36 20:46 (ARA) 20:53 (EPA) 22:0 22:111 22:46 22:56 22:53 22:63 (DHA) 24:19
0.067 ± 0.05 0.237 ± 0.108 0.113 ± 0.016 0.049 ± 0.004 0.662 ± 0.082 4.331 ± 0.525 0.318 ± 0.129 0.044 ± 0.003 0.050 ± 0.003 0.302 ± 0.067 0.260 ± 0.006 1.226 ± 0.184 1.776 ± 0.375 0.609 ± 0.069 1.203 ± 0.100 0.045 ± 0.016 0.062 ± 0.004 – 0.039 ± 0.007 0.240 ± 0.058 0.132 ± 0.026 0.178 ± 0.029 0.419 ± 0.033 0.414 ± 0.040 0.080 ± 0.004 – 0.147 ± 0.024 0.262 ± 0.018 0.524 ± 0.048 3.982 ± 0.224 0.176 ± 0.008
0.061 0.262 0.083 0.040 0.493 4.094 0.379 0.029 0.039 0.223 0.331 1.151 2.276 0.701 1.619 0.053 0.078 – 0.043 0.335 0.088 0.264 0.624 0.446 0.073 – 0.158 0.331 0.518 3.353 0.197
兺SAFA 兺MUFA 兺PUFA 兺FA
7.06 ± 0.87 3.53 ± 0.62 7.32 ± 0.38 17.91 ± 1.78
6.62 ± 0.65 4.19 ± 0.32 7.53 ± 0.14 18.34 ± 1.09
5.23 ± 0.26 2.71 ± 0.13 7.11 ± 0.30 15.05 ± 0.69
5.93 ± 0.17 3.81 ± 0.37 7.05 ± 0.12 16.79 ± 0.48
4.40 ± 0.06 2.54 ± 0.08 7.10 ± 0.38 14.04 ± 0.43
兺 3 兺 6 兺3/兺6 兺PUFA/兺FA Total FA
4.98 ± 0.29 2.34 ± 0.12 2.13 ± 0.07 0.41 ± 0.02 19.12 ± 1.90
4.39 ± 0.26 3.13 ± 0.19 1.41 ± 0.17 0.41 ± 0.02 19.49 ± 1.25
4.25 ± 0.34 2.86 ± 0.08 1.49 ± 0.15 0.47 ± 0.00 16.00 ± 0.74
4.13 ± 0.07 2.91 ± 0.06 1.42 ± 0.02 0.42 ± 0.02 17.83 ± 0.51
3.98 ± 0.28 3.12 ± 0.11 1.27 ± 0.04 0.51 ± 0.01 15.04 ± 0.51
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.014 0.048 0.014 0.007 0.100 0.360 0.064 0.007 0.005 0.049 0.014 0.100 0.177 0.055 0.102 0.004 0.010
± ± ± ± ± ± ±
0.003 0.025 0.043 0.033 0.024 0.017 0.004
± ± ± ± ±
0.003 0.005 0.011 0.257 0.002
0.043 0.131 0.063 0.032 0.395 3.226 0.149 0.025 0.041 0.184 0.321 0.880 1.415 0.505 1.394 0.054 0.500 – 1.037 0.189 0.131 0.228 0.599 0.490 0.064 – 0.143 0.309 0.488 3.225 0.206
SCEN ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.013 0.012 0.01 0.002 0.036 0.158 0.003 0.002 0.005 0.017 0.021 0.075 0.060 0.043 0.036 0.004 0.007
± ± ± ± ± ± ±
0.003 0.009 0.024 0.030 0.029 0.044 0.003
± ± ± ± ±
0.013 0.034 0.029 0.278 0.008
0.051 0.241 0.042 0.037 0.490 3.757 0.420 0.029 0.035 0.216 0.287 0.952 2.078 0.574 1.521 0.054 0.088 – 0.037 0.289 0.105 0.224 0.572 0.451 – – 0.142 0.298 0.468 3.124 0.181
UNFED ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.013 0.040 0.027 0.001 0.028 0.125 0.073 0.003 0.003 0.027 0.018 0.071 0.298 0.051 0.053 0.004 0.016
± ± ± ± ± ±
0.004 0.040 0.012 0.003 0.034 0.064
± ± ± ± ±
0.018 0.044 0.010 0.098 0.001
0.035 0.128 0.034 0.033 0.139 2.866 0.132 0.023 0.040 0.060 0.304 0.784 1.371 0.492 1.579 0.026 0.049 – 0.063 0.194 0.162 0.265 0.645 0.494 – – 0.148 0.296 0.478 2.957 0.221
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.007 0.021 0.018 0.008 0.063 0.081 0.012 0.001 0.004 0.032 0.016 0.058 0.020 0.022 0.057 0.005 0.010
± ± ± ± ± ±
0.010 0.014 0.013 0.020 0.102 0.031
± ± ± ± ±
0.012 0.046 0.016 0.245 0.020
– indicates not found or < 0.02 mg g −1.
Homogeneity of all group variances was tested by means of F max test (Fowler and Cohen 1996). All the groups gave lower ratios than the critical values of
0.05 level significance, except the two diet groups SAFA and MUFA. Normal distribution of the data was tested by checking if 70% of the parallel samples
174 Table 3. Effect of temperature on the fatty acid contents (mg g −1 dw), mean and standard deviation (n = 3), of Oreochromis niloticus dorsal muscle tissue. Total FA are calculated from the total integrated areas of all FA in the chromatograms. Fatty acid
16 °C
20 °C
25 °C (Control)
12:0 14:0 14:15 15:0 16:0I 16:0 16:17 17:0I 17:0 17:17 18:0I 18:0 18:19 18:17 18:26 (LA) 18:36 (GLA) 18:33 (ALA) 18:43 20:0 20:19 20:26 20:36 20:46 (ARA) 20:53 (EPA) 22:0 22:111 22:46 22:56 22:53 22:63 (DHA) 24:19
0.091 0.279 0.022 0.050 0.091 4.329 0.565 0.048 0.056 0.041 0.224 1.239 2.629 0.593 1.688 0.084 0.124 – 0.036 0.325 0.107 0.249 0.396 0.326 – – 0.190 0.312 0.633 4.351 0.148
0.051 ± 0.016 1.158 ± 0.347 0.055 ± 0.014 0.092 ± 0.009 0.171 ± 0.041 7.911 ± 0.805 1.990 ± 0.397 0.062 ± 0.002 0.070 ± 0.002 0.075 ± 0.017 0.284 ± 0.025 1.913 ± 0.079 7.081 ± 0.614 1.033 ± 0.084 2.742 ± 0.187 0.145 ± 0.019 0.280 ± 0.035 – 0.058 ± 0.000 0.761 ± 0.056 0.209 ± 0.046 0.290 ± 0.037 0.413 ± 0.043 0.345 ± 0.028 – – 0.184 ± 0.006 0.340 ± 0.032 0.699 ± 0.062 3.978 ± 0.247 0.126 ± 0.000
0.099 0.682 0.083 0.071 0.329 6.124 1.248 0.109 0.065 0.161 0.205 1.572 4.689 0.944 2.104 0.134 0.196 – 0.048 0.541 0.133 0.284 0.507 0.267 – – 0.235 0.546 0.503 3.694 0.143
兺SAFA 兺MUFA 兺PUFA 兺FA
6.35 ± 0.35 4.31 ± 0.45 8.36 ± 0.35 19.01 ± 0.77
11.64 ± 1.34 11.11 ± 1.00 9.56 ± 0.27 32.31 ± 2.07
9.30 ± 1.61 7.97 ± 1.86 8.60 ± 0.86 25.87 ± 4.32
兺 3 兺 6 兺3/兺6 兺PUFA/兺FA Total FA
5.44 ± 0.16 2.92 ± 0.25 1.83 ± 0.16 0.44 ± 0.02 20.17 ± 1.10
5.30 ± 0.30 4.26 ± 0.21 1.27 ± 0.12 0.30 ± 0.02 34.01 ± 1.92
4.66 ± 0.43 3.94 ± 0.43 1.18 ± 0.03 0.34 ± 0.02 27.49 ± 4.55
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.007 0.043 0.013 0.007 0.046 0.148 0.048 0.000 0.008 0.022 0.014 0.077 0.268 0.048 0.194 0.001 0.006
± ± ± ± ± ±
0.003 0.052 0.023 0.003 0.024 0.052
± ± ± ± ±
0.027 0.028 0.017 0.122 0.020
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.015 0.169 0.015 0.006 0.045 1.048 0.324 0.013 0.013 0.025 0.043 0.310 1.214 0.162 0.285 0.027 0.029
± ± ± ± ± ±
0.011 0.142 0.035 0.029 0.080 0.054
± ± ± ± ±
0.043 0.083 0.072 0.279 0.024
– indicates not found or < 0.02 mg g −1.
fell within the interval of the means ± SD (Fowler and Cohen 1996). All the data were approximately normal because two of the three parallels always fell within
the intervals (66%). Differences within the diet groups and within the temperature groups were tested by Tukey-Kramer ANOVA test (P = 0.05) which takes into account that several comparisons have been made, resulting in a risk for a type 1 error. Statistics were run using a JMP software package (SAS Institute Inc.).
Results The average total length of the different fish groups did not differ at the end of the experiment, whereas the mean weight was significantly higher in the control group compared to all treatment groups, with the exception of MICR1 (Figure 1, Final). There were no significant changes in average total length and total weight of fish fed on algae, and neither did the control group fed cichlid pellets show any significant increases in these averages (Figure 1). The control group also showed no significant increase in the condition factor (K). In contrast, all treatments showed significant decreases in K, except fish fed field-collected MICR1 (Figure 1). The unfed group showed the greatest decrease in K. Variations in total length and total weight of fish grown under different temperatures were also insignificant (Figure 2). Fish kept at 16 or 25 °C showed no significant increases in K, whereas those kept at 20 °C showed significant increases in K, 24% on average (Figure 2). The FA content of the pellets and algae used as diets in this study differed considerably among the feed types (Table 1). The pellets contained 4% EPA of 兺FA, small amounts of ARA, but no DHA, whereas all algae used in this experiment contained no ARA, EPA or DHA. The dominant FA in the blue-greens were palmitic (16:0) and ␥-linolenic acid (GLA, 18:36), but SPIR contained more GLA than palmitic acid. MICR1 (from the field) contained 3.8 times more ALA than MICR2 (grown in the laboratory). SCEN showed the highest FA content, with ALA completely dominating the FA (30% of 兺FA) (Table 1). Twenty-nine fatty acids of varying length and desaturations were identified in the treatment groups of fish (Tables 2 and 3). The most abundant FA in both the pellet fed and algae fed fish were palmitic acid, DHA, oleic acid (18:19), stearic acid (18:0), and LA. Other FA observed, but of minor importance, included vaccenic acid (18:17), ARA, EPA, and docosapentaenoic acid (22:53). The 兺FA, saturated
175 fed group (Figure 3). No significant differences were observed in the MUFA and PUFA analysed from the diet groups and the unfed group. The mean of 兺FA in the control group was nearly twice as much as that of the unfed group. SAFA was the only FA group that showed significant differences between some of the fish groups (MICR1 and MICR2) and the unfed group. The 3 PUFA was significantly higher in MICR1 than in the SCEN and STARV groups, whereas 6 PUFA varied both within the diet groups, and between the control and diet groups (Figure 3). DHA was significantly lower in the SPIR, SCEN and STARV groups than in the CONT and MICR1 groups and the 3/6 ratio was highest in MICR1 group and lowest in CONT group (Figure 3). Nearly all levels of the 兺FA, SAFA and MUFA were found to be highest in fish kept at 20 °C, intermediate in fish at 25 °C, and lowest in fish at 16 °C (Figures 4a, 4b). Both the SAFA and the MUFA were significantly higher at 20 °C vs. 16 °C, whereas PUFA did not vary significantly between the temperature groups (Table 4). At 20 and 25 °C, the SAFA were dominant, whereas at 16 °C the PUFA were more prominant (Table 3). Most of the individual FA showed patterns similar to 兺FA, SAFA, and MUFA, e.g., highest levels at 20 °C and lowest levels at 16 °C, e.g., 14:0, 16:0, 16:17, 18:19, and 20:19 (Table 3). The sum of 3 FA, and in particular DHA, decreased as temperature increased, whereas the sum of 6 FA did the reverse, i.e., increased with increasing temperature (Figures 4c, 4e). This resulted in a pronounced decrease in the 3/6 ratio (P = 0.0005) with increasing temperature (Table 4, Figure 4f). The levels of ALA and LA were lowest at 16 °C whereas EPA and ARA were low and no significant differences were observed when data from the three temperatures were compared (Figures 4d, 4e). The ratio PUFA/ 兺FA was low (< 0.5) and did not vary much within the temperature range (Figure 4f).
Discussion Figure 2. Change in total length (a), total weight (b), and condition factor (c) of O. niloticus grown at three temperature regimes. Bars indicate SD of the mean. No bars indicate that SD is smaller than the dots.
fatty acids (SAFA) and monounsaturated fatty acids (MUFA) were significantly higher in the control fish group than in the diet fish groups, including the un-
Although no significant changes in fish length and weight were observed in the algal treatments, we found that the FA contents of the food had slightly influenced the FA content of the dorsal muscle tissue. This became evident when similarities between the major FA of the diet were compared to the FA of the muscle tissue. The dominance of palmitic acid, oleic acid, and linoleic acid (LA) in the tissue lipids indi-
176
Figure 3. Major fatty acid content (mg g −1 dw) of Oreochromis niloticus muscle tissue fed pellets and different algal diets (see the legends of Figure 1). Bars indicate SD of the mean. No bars indicate that SD is too small to be shown. Columns, which do not share common letters, are significantly different at P ⭐ 0.05, based on Tukey-Kramer’s test.
cate the deposition of these FA from the algal diets, which are rich in the above mentioned FA (Tables 1,
2 and 3). However, direct comparison of individual fatty acids was a difficult task, particularly regarding
177
Figure 4. Contents of major fatty acid groups of Oreochromis niloticus tissue grown at 3 different temperatures. Vertical bars indicate SD of the mean. No bars indicate that SD is smaller than the dots.
PUFA which were very similar in all the fish groups. The dominance of long-chained PUFA in the fish tissue, particularly DHA which was absent in both the algal and commercial diets (the control), indicates that the elongation and desaturation enzymes were
very effective in the herbivorous Oreochromis niloticus. We also suggest that in fish adapted to warm waters, such as Oreochromis, temperature may not be the overriding factor influencing the fatty acid content for at least two reasons. Firstly, this fish can not
178 Table 4. ANOVA test (Tukey-Kramer HSD) on groups and single fatty acid content (FA) of Oreochromis niloticus at three different temperatures, 16, 20 and 25 °C. FA groups
df
F-value
P-value
Individual effects of the treatments
兺FA SAFA MUFA PUFA 兺 3 兺 6 3/6 ALA LA EPA DHA ARA
2 2 2 2 2 2 2 2 2 2 2 2
16.91 14.06 22.40 3.90 5.16 15.05 35.89 26.23 16.50 2.35 6.35 3.60
0.0034 0.0054 0.0016 0.0821 0.0497 0.0046 0.0005 0.0011 0.0036 0.1759 0.0330 0.0939
20 ⬇ 25, 25 ⬇ 16, 20 > 16 20 ⬇ 25, 25 ⬇ 16, 20 > 16 20 > 25 > 16 20 ⬇ 16 ⬇ 25 16 ⬇ 20 ⬇ 25 20 ⬇ 25 > 16 16 > 20 ⬇ 25 20 > 25 > 16 20 > 25 ⬇ 16 20 ⬇ 16 ⬇ 25 16 ⬇ 20, 20 ⬇ 25, 16 > 25 25 ⬇ 20 ⬇ 16
survive the low temperature (5 °C) which is supposed to bring about an increase in PUFA. Secondly, Oreochromis was capable of maintaining its PUFA level, irrespective of the tested temperatures. On the other hand, in order to survive at sub-optimal temperatures (16 °C) this fish seems to need more 3 FA (mainly DHA) than 6 FA. Fish supplied with algae, excepting MICR1, did not increase in length; instead, they seemed to lose weight just as the unfed group did (Figure 1). Unfortunately, the fish sizes varied a bit at the start of the experiment, because they had been kept four months in the laboratory before the experiment could be initiated. In addition, some fish grow much faster than others in the same brood, this being due to dominance behaviour, a well-known phenomenon in fish cultures. This effect was quite evident in the control group, where some fish grew large and dominated the smaller ones that seldom succeeded in getting food at feeding time. The fish were not tagged, so it was also impossible to follow the growth of individual fish. Therefore, the changes in the condition factor (K) were calculated, rather than actual lengths and sizes (Figure 1c). The K-values agree very well with earlier data from lakes in Ethiopia: K = 1.4–2.4 in L. Tana (Zenebe 1997), 1.9–2.3 in L. Chamo, and 1.6– 1.8 in L. Langano (Zenebe 1998c). As expected, no significant change in K was observed in the control since the fish were fed the same cichlid pellet diet both before and during the feeding experiments. The significant decrease in the condition of the fish in all treatments, except MICR1, can be explained by both the quality and quantity of the algal feed provided to
the fish. The amount of algae (2% of body weight) fed to the fish in the present study may not have been sufficient to induce fish growth. In particular, the small, single-celled MICR2 and SCEN were probably difficult for the fish to collect and may not have been completely ingested (Northcott et al. 1991; Dempster et al. 1995). The helicoidal SPIR threads, on the other hand, were observed to be easily collected by the fish. The dominant fish in the control group supplied with cichlid pellets grew very well because the pellets were big enough to be detected and all were ingested (cf. the SD bars in Figure 1b). Moreover, the pellets are a high quality feed and as such, are nutritionally balanced. The pellets also contained a small amount of EPA (4%), which is important to the growth of juvenile fish (Table 1) (Watanabe et al. 1982; D’Abramo and Sheen 1993). The explanation for why fish fed on MICR1 did not lose condition, as the other treatments did, might be that some Microcystis colonies, besides being easy for the fish to collect, occasionally contained traces of diatoms and cladocerans. A diet of mixed algae is qualitatively superior in nutrients than a monoalgal diet (Lobel 1981; Lundstedt and Brett 1991). However, during microscopic examination of the collected samples, single zooplankton was noted only in one of the samples. Also, no EPA or DHA were found in the FA data analysed from a mixture of all MICR1 samples collected (Table 1). Therefore, a more likely reason for the stability in K could be that MICR1 contained nearly four times more ALA than MICR2, which was the highest level of DHA traced and lead to the highest 3/6 ratio of 2.1 in the MICR1 fed
179 fish (Table 2, Figure 3). Both 3 and 6 FA are important to fish, but in general, fish have higher requirements for 3 than for 6 FA, which is quite the opposite of terrestrial mammals (Sargent et al. 1995). The 3 PUFA series are also selected over the 6 series as substrate for the desaturating enzymes (Sargent et al. 1995). On the other hand, SCEN contained very high levels of ALA (35% of 兺FA), although this was not mirrored in the low 3/6 ratio in the fish (Table 2). This may indicate that this single-celled alga was too small for the fish to efficiently ingest. In both the Microcystis aeruginosa species (MICR1 & MICR2), there were higher levels of GLA than of ALA (3/6 ratios < 1, Table 1). These levels and ratios are consistent with published data on these species, both from cultures (Kenyon 1972; Piorreck et al. 1984) and from field samples from L. Langano with 90% Microcystis (Zenebe et al. 1998a), but are inconsistent with data from other clones and temperate lakes (3/6 ratios from 1.5–2.3, Ahlgren et al. (1992)). However, the laboratory grown MICR2 contained more than twice the GLA of the field collected MICR1, which is most likely the result of the high incubation temperature (25 °C) that has been shown to favour the production of GLA (James et al. 1989). Thus, MICR2 may have been very similar to SPIR, which is characterised by high levels of GLA and slight traces of 3 FA (Table 1), or it could also have been completely lacking in 3 FA (Cohen 1987; Ahlgren et al. 1992). It is unknown whether GLA is utilized by fish. However, there is documentation which indicates that Oreochromis niloticus is capable of elongating and desaturating LA into ARA, and ALA into EPA and DHA (Olsen et al. 1990). Hence, since the same desaturases are used in both the 3 and 6 families (Sargent et al. 1995), it is likely that Oreochromis niloticus is also capable of utilizing GLA, which is an intermediate in the elongation and desaturation route in the production of ARA (Sargent et al. 1995; Ahlgren et al. 1999). In support of this hypothesis, we found that ARA was higher in the MICR2 and SPIR fed fish compared to the MICR1 fed fish (Table 2). Thus, all algal fed groups, except MICR1, decreased in K as did the unfed group, but probably for different reasons. Very low 3 FA in SPIR, i.e., low food quality, was probably the main reason for the decrease in K in the fish group, whereas low quantity, depending on the difficulty collecting such small cells, were a more probable cause in the SCEN fed fish. In filter-feeding experiments, it has also been
shown that green algal cells are less digestible (45%) to tilapia than are cyanobacteria (76%), probably due to the hard cell walls (Dempster et al. 1995). Concerning the decrease in K observed in the MICR2 fed fish, both the quantity and quality of the algal food might have been inadequate for fish growth. In a bioenergetic model based on literature data of ingestion rates, Dempster et al. (1995) predicted that filterfeeding on small algal cells fails to support growth in tilapias due to the resulting negative energy balance. Our results support this prediction, showing that a mixed diet (pellets) is superior as food even to herbivorous tilapias than separate algal diets. In our field study, we also found that both food quality and quantity are important to tilapia growth and development (Zenebe et al. 1998a). The condition and performance of Oreochromis niloticus was found to be extremely variable from lake to lake, depending on the type of food items available to the fish. However, the range of variation observed in the present study is much less than that observed in our field study. This difference in variation is probably due to the fact that the algal diets used in the present study are more similar to each other than were the natural phytoplankton communities of the lakes studied in the field (Zenebe et al. 1998a). Another plausible explanation may be a genetic uniformity of this cultured fish vs. that of the feral fish from the earlier study. Generally, an increase in temperature will increase the growth rate of tilapia. In the present study, the higher K observed for the fish at 25 and 20 °C vs. that at 16 °C confirms the role of temperature, since all were fed the same pellet food (Figure 2). This result was also confirmed in an aquaria growth experiment conducted concurrently with this study (Admassu 1998). Temperature influences both the feeding rate and conversion efficiency of the feed (Caulton 1982). Experiments have shown that the optimal water temperature for the growth of tilapias is about 30 °C (Cridland 1961; Caulton 1978). We have also observed that in lakes with high water temperature, such as Lake Chamo (> 25 °C), fish grew much faster than in cooler Rift Valley Lakes of Ethiopia (< 25 °C, e.g., Lake Awassa and Langeno) (Getachew (1993) and Zenebe et al. (1998a, 1998b), D. Admassu, pers. comm.). On the other hand, fish kept at 16 °C did not grow at all, which was very likely due to the low feeding rate of the fish at this low temperature (Figure 2). At 16 °C, the fish ate very seldom. Their behavior was similar to that of the unfed fish, i.e., they
180 spent most of their time at the bottom of the aquarium and seldom moved. In contrast, at 25 °C, the fish were very active and nearly jumped out of the aquaria at feeding time, whereas the fish at 20 °C showed more moderate movement. These observations agree with earlier findings that tilapia generally ceases feeding behavior when the temperature drops below 16 °C (Chervinski 1982). At 20 °C, the fish stored more fat (SAFA and MUFA) than fish kept at 25 °C (Figure 4). This is because fish expend more metabolic energy at 25 °C than at 20 °C (Caulton 1982). The lowest SAFA and MUFA contents occurred at 16 °C and is again very likely the result of starvation due to the low feeding rates observed at this low temperature (Caulton 1982; Chervinski 1982). In contrast, the level of PUFA showed no significant variation between the different temperature groups (Table 4). Thus, temperature had little influence on the PUFA content of Oreochromis, which is a thermophilic fish. This finding contradicts the Farkas (1984) observation of an increase in the level of liver PUFA in Cyprinus carpio, kept at the low temperature of 5 °C. However, the PUFA consists of two groups, 3 and 6 FA, each with different functions, as well as relationships which varied depending upon the temperature. At 16 °C, the 3 FA were slightly higher (significant at 90% level) and the 6 FA were significantly lower (Table 4, Figure 4c), resulting in a high 3/6 ratio (1.8) when compared to the results from 20 and 25 °C (3/6 = 1.3 and 1.2, respectively). Considering the 3 and 6 PUFA in more detail reveals some interesting differences between the 18 carbon PUFA and 20–22 carbon PUFA: The levels of both LA and ALA were lowest at 16 °C (LA significantly lower than at 20 °C) whereas ARA showed no difference. In contrast, DHA was highest at 16 °C (significantly higher than at 25 °C) (Table 4, Figure 4e). That might indicate that the herbivorous fish could selectively modify the 3 desaturation route differently from the 6 route (cf. Ahlgren et al. (1999)). Comparing the seldom eating group at 16 °C (Table 3) with the unfed group at 25 °C (Table 2) also shows some interesting differences. All the groups’ SAFA, MUFA and PUFA, including the 3 PUFA showed higher levels in the fish group at 16 °C than the unfed group, whereas the 6 group did not differ. A lower respiration rate at 16 °C is probably the main reason for this result. The 3/6 ratio was also much higher in the fish group grown at 16 °C. It seems as if 3 FA is more important than 6 FA at sub-opti-
mal temperatures. Specifically, the 3 FA known as DHA was mainly responsible for this difference. DHA is an important substance in neural tissue and in the retinal membranes in the eyes of fish (Sargent et al. 1995), whereas 6 PUFA including ARA are the precursors for the eicosanoids (biologically active metabolites) which regulate protein synthesis (Bell et al. 1991). At temperatures far below optimum, fish probably do not invest in tissue growth and reproduction processes, concentrating instead on keeping neural tissue intact. Under these conditions, the fish are apparently capable of selective production of DHA, at the expense of ARA, primarily in order to support vital tissues, i.e., supporting neural tissue over muscle tissue.
Summary and conclusion The fatty acid content of food mainly influenced the fat stores, SAFA and MUFA, in the fish dorsal muscle. In addition, during starvation, SAFA and MUFA were utilised by the fish, but the PUFA were not. To some extent, the 18 carbon 3 and 6 PUFA in the food items could be traced in the long-chained derivatives, e.g., ARA in the MICR2 and SPIR fed fish and DHA in the MICR1 fed fish. However, two of the algae in the diets, MICR2 and SCEN, given in the same quantities as the control pellets, were probably too small in size to be efficiently grazed. Oreochromis seems to have a rather great capacity to modify FA, found in algal food, into their own species specific FA patterns. Therefore, the observed influence appears to stem from both the amount and the type of food. On the other hand, we cannot expect large differences in the fish, since the MICR2 and SPIR used in the present study showed more similarities in their FA contents than expected; however, all the algae lacked ARA, EPA and DHA. In contrast, the cichlid pellets contained some EPA and traces of ARA and are, therefore, a superior food for Oreochromis. Dietary effects may be better demonstrated by using other groups of algae, including diatoms and flagellates, which are easily digestible and rich in long-chained PUFA. Unfortunately, we were unable to grow these algae in the quantities required for this feeding experiment. The decline in SAFA and MUFA as temperature decreased was very likely due to reduced feeding rates, which suggests that the observed effect of temperature on the FA content of Oreochromis niloticus
181 is an indirect one, i.e., the direct influence of temperature was to change the feeding rate of the fish. Oreochromis is adapted to warm waters. High water temperature promotes high feeding rates, high conversion efficiency and better growth of the fish. However, the levels of DHA showed an inverted relationship to temperature, with the highest 3/6 ratio at the lowest temperature. This fish apparently has a great capacity for selective accumulation of certain 3 PUFA (DHA) at the expense of the 6 PUFA (ARA) required for survival at suboptimal temperatures.
Acknowledgements We are grateful to the staff of the Department of Biological Sciences, University of Stirling for their scholarly suggestion on the set-up of our experiment and also for providing us with juvenile tilapia. We also thank Siv Tengblad for performing the FA analyses, Yosef Tekle-Giorgis for advice on some of the statistics, and two unknown referees whose critiques and suggestions improved an early version of this paper. We acknowledge the financial and material support given by the Swedish Agency for Research Co-operation with Developing Countries (SAREC) to the School of Graduate Studies of the Addis Ababa University (AAU).
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