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The use of silage made from fish and abalone viscera as an ingredient in abalone feed
Aqllzhcumlre ELSEVIER
Aquaculture 140 (1996) 87-98
The use of silage made from fish and abalone viscera as an ingredient in abalone feed Maria Teresa Viana *, Lus M. L6pez, Zaul Garcia-Esquivel, Elda Mendez Institute de Investigaciones Ocean&gicas,
Universidad Aut&oma de Baja California, PO Box 4.53, 22 800 Ensenada, BC. M&co
Abstract Silage prEpared from fish and abalone viscera are effective dietary protein sources for the juvenile abalone, Hal&is filgens. Significantly higher growth rates occurred when abalone were fed artificial diets containing heated fish silage (53 p,rn day-‘) and unheated fish silage (61 p,m day- ‘) as a protein source compared with the kelp, Mucrocystis pyrifera (1.5 pm day- ‘>. However, no differences were found between diets containing heated and unheated fish silage at 30% protein inclusion, suggesting that the degree of hydrolysis did not affect protein utilization by abalone. Similar results were obtained when abalone viscera silage was used (50 p,m day-‘), producing faster growth rates than kelp (18 pm day-‘) or kelp meal (12 p,m day-‘). In the same experiment a significant increase in growth rate was observed when abalone with low growth rates, resulting from feeding on kelp and kelp meal, were switched to a diet containing abalone viscera silage. These animals exhibited higher growth rates (135 and 167 km day-‘) than animals fed this diet throughout the trial (122 p,rn day-‘), suggesting the presence of compensatory growth of organisms. Keywords: Abalone; Artificial diets; Diets; Feed; Growth; Protein
1. Introduction Artificial diets important as an Sorgeloos, 1992; plates containing organisms reach
based on less expensive protein sources are becoming increasingly alternative to live feeds in the aquaculture industry (Coutteau and Curatolo et al., 1993). Recently set abalone spat are usually reared on diatom films as feed and weaned to fresh macroalgal diets when the a size of about 3-4 mm (Hahn, 1989). Nevertheless, it has been
* Corresponding author. 00448486/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0044-8486(95)01 196-X
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documented that H. discus, H. discus hannai and H. sieboldii fed with various artificial diets grow faster than those fed with natural algae (Nie et al., 1986; Hahn, 1989). The faster growth rates were attributed to the higher protein content and nutritional quality of the feed. Research conducted by Uki et al. (1985) showed that abalone growth rate was faster when fed a diet supplemented with casein compared with other protein sources. Clearly, casein was used more efficiently by abalone. Thus, inexpensive, high quality protein is an essential element for profitability in the aquaculture industry. Siiage has proved to be a good protein source for sea-farmed fish (Raa and Gildberg, 1982). Fish silage preserved by adding acid (a combination of organic and inorganic acids) is acceptable to salmonids and the nutritional value is as good as that of the fresh, raw material (Austreng, 1982). However, the preservation process causes fish silage to become liquefied, and hence large volumes of dry binder meals are needed to produce a moist pellet of acceptable consistency, resulting in formulation problems. In such a moist feed, 60-70% of the dry matter may result from the added dry meal (Raa and Gildberg, 1982; Raa et al., 1983). During prolonged storage, large amounts of free amino acids may be present in silages owing to the activity of endogenous enzymes in fish tissues. These enzymes are mostly proteases, such as pepsin-type stomach proteases, and lysosomal enzymes, like cathepsin D, which is the major muscle protease (Gildberg, 1982; Gildberg, 1988). Extensive hydrolysis may result in a reduction of the nutritional value of silage. When mixtures of free amino acids are fed, only the absorption mechanism for free amino acids is utilized. As a result, more of the ingested amino acids may be lost in the faeces due to increased competition between amino acid carriers (Espe, 1993). In addition, large amounts of free amino acids present in the pellets may augment protein leaching. It has been shown that partially hydrolysed material is superior in nutritional value to fully hydrolysed material (Stone et al., 1989; Espe et al., 1993). In order to avoid excessive hydrolysis during prolonged storage, enzyme activity from the proteases present in fish silage can be inactivated by heating the raw fish material for 5 min at 60°C prior to ensilaging (Viana et al., 1993b). This results in a 15% reduction in free amino acid levels after 90 days compared with unheated silage. The use of fish and abalone viscera silage as alternative protein sources for abalone could result in cheaper artificial feeds, making their culture more economically viable. The aim of the present study was to test whether diets made with abalone viscera silage and heated and unheated fish silage from mackerel (Scomber juponicus) had a significant effect on the growth rates of the juvenile abalone, H. fulgens. 2. Material and methods 2.1. Diet preparation Silage was made as described by Viana et al. (1993b). In summary, heated (at 60°C for 5 min) and unheated mackerel, S. juponicus (Experiment 1) and heated (at 60°C for 5 min) abalone viscera (Experiment 2) were mixed, when cold, with 2.6% phosphoric acid, 2.6% citric acid and 0.1% sodium benzoate as a preservative. The mixtures were blended to obtain a homogenate and left for 60 days in plastic buckets.
M.T. Viana et al./Aquaculture Table 1 Percentage
composition
Ingredients
of the four artificial Experiment Heated fish silage diet
Silage a Fish meal b Soy bean meal ’ Corn meal d Starch (rice) Kelp meal e Sodium alginate ’ Gelatin (50 blooms) Vitamin mixture g Mineral mixture s Cellulose Methionine
140 (1996) 87-98
89
diets tested, given as a percentage
1
Experiment
of dry matter 2
Abalone viscera silage diet (AS)
Kelp meal diet (KM)
(HS)
Unheated fish silage diet (US)
31.8 0 5 15 10 10 10 6 2 5 5 0.2
31.8 0 5 15 10 10 10 6 2 5 5 0.2
20 16.8 10 10 10 10 10 6 2 5 0 0.2
0 0 0 0 0 82 10 6 2 0 0 0
a Mackerel were kindly supplied by Tecmar, SA, and abalone viscera by the fishery cooperative Bahia Tortugas, BCS.b Supplied by Productos de Ensenada, SA.C 50% protein, kindly supplied by the American Soybean Association.d Mazeca, produced in Mexico? Kindly supplied by CRIP (Centro Regional de Investigaciones Pesqueras, Mexico).’ Kelgin MV, kindly supplied by KeIco.g As recommended by Hahn (1989), using Stay-C from Roche.
Silage diet formulations (Table 1) were based on the constituents recommended by Uki et al. (1985) and Uki and Watanabe (1992). Vitamin and mineral mixtures were those recommended by Hahn (1989). All ingredients were mixed until a complete homogenate was obtained. The diet was then rolled flat to a thickness of 2 mm. Pieces 2 cm x 2 cm were cut for Experiment 1, and pieces 1 cm X 1 cm for Experiment 2. They were then frozen at - 20°C until used. The fish silage pellets used in Experiment 1 were tested for their stability in seawater, both in terms of dry matter and protein. To determine dry matter stability, four pellets of each diet were placed in three control buckets and their dry matter loss was measured after 12 h at 20°C. In addition, a control bucket without animals was used for each diet during Experiments 1 and 2 to measured the daily dry matter loss throughout the experiment. Protein leaching was determined at various temperatures by immersing pellets in seawater at constant turbulence and at 16, 20 and 24°C. A 1 ml sample was taken from the remaining material at 2, 4, 6 and 12 h and its soluble protein content was determined using the Lowry method (Lowry et al., 1951). As enzyme activity is expected in the unheated silage (Gildberg, 19821, the activity present in the fish silage diets was tested. Enzymatic activity was measured at pH 3 following the method of Barret (1972). The incubation mixture consisted of 0.5 ml Johnson/Lindsay buffer (Johnson and Lindsay, 19391, 0.25 ml sample extract and 0.25 ml 8% haemoglobin. The concentration of folin-positive material in the supematant, after adding trichloroacetic acid (TCA, 5% end concentration), was determined by
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140 (1996) 87-98
Lowry’s method (Lowry et al., 1951) using tyrosine as a standard. Enzyme activity was also measured at pH 5, which is typical of abalone stomach pH (determined from 20 animals collected from the sea). Azocasein was used as a substrate. Azocasein solution (0.25 ml of a 6% solution dissolved in distilled water) was incubated with 0.65 ml citrate-phosphate buffer and 0.1 ml pellet extract. After 40 min at 37”C, the reaction was stopped by the addition of 3 ml of 3% TCA. The mixture was filtered and the amount of trichloroacetic acid-soluble peptides was measured at 360 nm. To assess the dietary value of the artificial diets, a control diet of fresh kelp (K) was used in Experiments 1 and 2 and an artificial diet made from kelp meal (M. pyriferu) (KM) was used in Experiment 2. Pellets were made by adding 27% water to a mixture of gelatin, sodium alginate, vitamins and dried kelp meal (Table 1). 2.2. Proximate analysis Percentage dry weight was calculated as the dried residue weight of triplicate samples (4-5 g> of each diet after being dried to constant weight at 100°C. Total nitrogen was determined using triplicate samples analyzed by the Kjeldahl method (AOAC, 1990). Crude protein was calculated as %N X 6.25. Total lipid was determined using triplicate samples (10 g) by a column procedure utilizing methanol-chloroform/water as the eluting solvent (Bligh and Dyer, 1959). Ash was determined by heating duplicate samples (3 g) of each diet to 600°C for 18 h. 2.3. Experimental procedure 2.3.1. Experiment 1 Six-month-old H. $&ens with an average shell length of 18.3 mm (SE 0.66) and average weight of 0.6 g (SE 0.06) were obtained from the laboratory of Bahia Tortugas BCS. The abalone were held in flow-through, aerated seawater with a controlled flow of 300 ml min- ‘. Daily average temperatures ranged from 20 to 23°C throughout the feeding trial. Oxygen, pH and ammonium content were monitored (data not shown). Feeding experiments were conducted after 21 days of acclimation to laboratory conditions. During this period abalone were fed fresh M. pyriferu. The effects on growth rate of the artificial diets containing unheated (raw) mackerel silage (US) and heated mackerel silage (HS) were compared with those on a control diet of the fresh kelp, M. pyriyeru (K). Animals were held in 20 1 plastic containers (three replicates per treatment) with 32 abalone per container. Animals were marked with plastic tags so that individual growth could be monitored. Diets were offered for 12 h overnight (ad libitum) every night for 25 weeks. Any remaining food was collected daily. An attempt was made to estimate the food conversion efficiency, but the results were unreliable because of inaccurate recovery of the uneaten pellets. Twice a week algae growing on the inside walls of the containers were removed with a soft brush. Whole-body weight was measured monthly with an electronic balance (0.001 error) and shell length with electronic digital calipers (fO.O1 pm). Specific growth rate (SGR, % day- ’ > was estimated according to Espe (1993): SGR = lOO{(In final weight - In start weight) /days of experiment}
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140 (1996) 87-98
91
2.3.2. Experiment 2 Two-month-old abalone with an average shell length of 8.9 mm (SE 0.93) and weight of 0.1 g (SE 0.01) were obtained from the laboratory of Bahia Tortugas BCS and held in the same conditions as in Experiment 1. Feeding experiments were conducted using four replicate groups of 32 abalone for each diet treatment. Animals were acclimatised to laboratory conditions for 35 days and fed an artificial diet containing abalone viscera silage. Daily average temperature varied from 21 to 24°C throughout the experimental trial. During the experiment, abalone were fed ad libitum for 12 h overnight for 8 weeks. One group was fed the fresh kelp, M. pyrijeru (K), the second was offered the kelp meal diet (KM) and the third the artificial diet containing heated abalone viscera silage (AS) as a protein source. After the 8th week all animals were transferred to the AS diet for an additional 7 weeks. During this time, no container replication was used and food was offered to satiation 24 h per day. Twice a week, all algae growing on the inside walls of the containers were removed with a soft brush. Whole-body weight was measured monthly with an electronic balance (0.001 error) and shell length with digital calipers. 2.4. Statistical
analyses
In Experiment 1, a one-way ANOVA or a two-sample t-test (Zar, 1984) was used at specific times in order to compare mean specific growth rates (SGR) across diet treatments. A one-way repeated ANOVA compared SGR over time, followed by a multiple comparison of the means (SNK, Student-Newman-Keuls test). Shell length was compared over time with a repeated ANOVA test, while comparison across diet treatments was performed with a Kruskal-Wallis range test, followed by non-parametric Dunn’s multiple comparisons (Zar, 1984) owing to violations of homoscedasticity in the raw data. A two-way ANOVA test compared SGR of abalone across diet and time treatments during the first 8 weeks of Experiment 2 (3 diets X 2 times X 4 replicates). The ANOVA test was followed by multiple comparisons of the means (SNK test) to identify the groups which differed. Since no replicate containers were used in the second half of the experiment, a two-way ANOVA test without replication was used to compare SGR between diets (3 diets X 2 times). The computer package of Sigma-Stat for DOS was used in these statistical analyses.
3. Results 3.1. Experiment
I
The fish silage diets were similar in protein (31-33%) and lipid (3-5%) while the fresh kelp diet was lower in both protein (2%) and lipid (0.1%) (Table pellet stability test indicated a dry matter loss of 10.0% (SE 0.4) for the HS 2.6% (SE 0.3) for the US diet after 12 h submersion at 20°C. Significantly (P < 0.05) mean protein leaching occurred at 20 and 24°C compared with 16°C
content, 2). The diet and greater for both
M.T. Viana et al./Aquaculture
92 Table 2 Proximate
analysis
of the diets used in Experiments
140 (1996) 87-98
1 and 2, expresed as a percentage
Diet
Crude protein
Total lipid
Ash
Water
Heated fish silage (HS) Unheated fish silage (US) Abalone viscera silage (AS) Kelp meal (KM) Fresh kelp (K)
33.1 31.9 31.2 5.1 2.0
3.8 3.3 4.7 0.3 0.1
18.2 16.1 18.9 14.1 12.0
24.1 29.6 46.6 31.0 84.0
the US and HS diets. No significant differences (P > 0.05) occurred between the US and HS diets at 20 and 24°C. However, the US diet incubated at 16°C exhibited significantly greater (P < 0.05) mean protein leaching than the HS diet (98.7 vs. 90.1 mg g-l protein, respectively). At pH 3, the HS diet showed a remaining enzyme activity of 0.36 krnol tyr eq ml-’ h- ’ compared with 2.15 for the US diet. No activity was registered when pellets were assayed at pH 5. Abalone fed silage diets exhibited significantly different (P < 0.05) shell growth rates over time (F’s = 55.1 and F,*s = 45.2 for HS and US diets, respectively) (Fig. 1, Table 3). During the first month abalone growth was slow (3.0 and 2.0 pm day-’ on the HS and US diets, respectively), but gradually increased to 46-65 pm day- ’ (average 53 km day-’ ) on the HS diet and between 52 and 67 pm day-’ (average 61 p,rn day-‘) on the US diet. Animals fed the control diet K grew at significantly lower (P < 0.05) growth rates during the first 2 months (l-2 pm day-‘), and subsequently died (Fig. 1, Table 3). Significantly different shell growth rates (Kruskal-Wallis test, H = 30.15, df = 2, P < 0.001) occurred between diet treatments after the second month (end of kelp treatment), but no differences were detected between animals fed silage-based diets at
30 26 26E g
24-
r_ p
22-
u = a,
20 -
l
’ .
Heated silage diet (HS) Unheated
silage diet (US)
Fresh kelp (K)
??!I 16 -
0
5
10
15
20
25
30
Time (weeks) Fig. 1. Experiment 1. Shell length (mm) of abalone fed with heated (HS) and unheated and fresh kelp (K). Means and standard errors are given.
(US) mackerel
silage
M.T. Viana et al,/Aquacubure
I40 (1996) 87-98
93
Table 3 Growth rates of juvenile abalone fed for 7 months on artificial diets containing heated mackerel unheated silage (US) and a natural diet of fresh kelp (K). Standard errors in parentheses Diet
No. of weeks
HS
0 4 8 13 17 22 25
us
K
silage (HS),
Growth rate (km day-‘)
(% day- ‘)
3.1 34.4 46.2 55.1 47.1 64.2
0.5 (0.1) 0.7 (0.3) 0.9 (0.1) 0.8 (0.1) 0.9 (0.1) l.O(O.1)
(1.2) (3.2) (4.1) (3.1) (2.6) (3.9)
0
4 8 13 17 22 25
1.6 (0.3) 22.3 (3.5) 51.5 (4.1) 63.5 (7.3) 61.5 (7.1) 67.4 (4.7)
0.5 0.7 1.0 0.8 0.9 0.8
0 4 8
1.2(0.5) 2.4 (0.8)
0.2 (0.1) -0.3 (0.1)
(0.1) (0.1) (0.1) (0.1) (0.1) (0.1)
this time (Dunn’s multiple comparison, P < 0.05) or by the end of the experiment (two-sample test, t = 0.523, df = 50, P = 0.603) (Table 3). Body weight gain followed a similar pattern to shell length (Fig. 2). The one-way ANOVA indicated significantly lower SGRs for abalone fed the K diet during the first (F2,63 = 16.3; P < O.OOl>and second (F2v63= 82.1; P < 0.001) month of the experiment compared with those offered silage diets (Table 3). Animals fed the K 2.4
-
2.0 -
l
Heated silage (HS)
l
Unheated
A
Fresh kelp (K)
silage (US)
s E .P
1.6 -
g &
1.2 -
$
0
5
10
15
20
25
30
Time (weeks) Fig. 2. Experiment 1. Body weight (g) of abalone fed with heated (HS) and unheated (US) mackerel silage and fresh kelp (K). Means and standard errors are given.
M.T. Viana et al./Aquaculture
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140 (1996) 87-98
diet exhibited a negative SGR during the second month ( - 0.3% day- ’ >, just prior to their death. The SGRs of animals feeding on the silage diets were low for the first month (0.5% day-’ ), but subsequently maintained a constant SGR of 0.9% day-’ until the end of the trial (Table 3). 3.2. Experiment
2
The mean shell length of abalone was significantly different between diets (F2 = 38.1, P < 0.001) and between times (Fz = 53.6, P < 0.001; Fig. 3). A significant interaction
effect between time and diet was observed during the first half of the experiment (F4 = 12.6, P < C.OOl), owing to the sustained shell growth of animals fed the AS diet (average 50 pm day-’ > in contrast to the halt in growth of animals feeding on the KM and K algal diets (Fig. 3). A change in shell pigmentation was associated with the diets. The AS diet produced an intense blue-coloured shell, while the KM and K diets produced a light blue shell. Once all animals were switched to the AS diet, growth rates in terms of length increased markedly (Table 4). The increase in growth rate at this time (average 122 pm day-‘) by the animals fed the AS diet throughout suggests that changes in the experimental procedure influenced the results. Nevertheless, at the end of the experiment animals previously fed the KM diet were similar in length to those fed the AS diet throughout (Fig. 3). Body weight gain followed a similar pattern to shell length (Fig. 4). SGRs decreased during the first half of the experiment and subsequently recovered during the last 8 weeks (Table 4). A two-way ANOVA test indicated highly significant effects of diet (Fz,,8 = 104.6, P < 0.001) and time factors (F,,,s = 169.4, P < 0.001) during the first half of the experiment, with higher values observed for animals fed the AS diet, followed by those fed KM and K (Table 4). A significant interaction term between diet and time treatments was also found (F2.t8 = 8.2, P < 0.003). Both groups
20 16 -
E
16-
z. 5
14-
@ 3 z
12 -
l
Abalone silage diet (AS)
.
Kelp meal diet (KM)
A
Fresh kelp(K)
5 10 -
0I
I
I
I
I
I
I
I
0
2
4
6
6
10
12
14
Time (weeks) Fig. 3. Experiment 2. Shell length (mm) of abalone fed with artificial diets containing heated abalone viscera silage (AS), kelp meal (KM) and a natural diet of fresh kelp (K). Means and standard errors are given. Arrow indicates a shift to AS diet.
M.T. Viana et al./Aqunculture
I40 (1996) 87-98
95
Table 4 Growth rates of juvenile abalone fed for 8 weeks on artificial diets containing abalone viscera silage (AS), kelp meal (KM) and a natural diet of fresh kelp (K). The KM and K treatments were switched to the AS diet after the 8th week. Standard errors in parentheses Diet
AS
KM
Diet change +
K
Diet change --t
’ Change of experimental
No. of weeks
Growth rate
(km day- ’ )
(% day- ‘)
0 4 8 12 a 15
64.9 (1.8) 34.6 ( 14.7) 132.9 a 111.4
2.2 (0.1) 1.4(0.1) 3.3 a 2.1
0 4 8 12 15
30.4C1.5) 5.6 (4.1) 155.7 177.1
1.1 (0.1) 0.1 (0.1) 4.5 4.0
0
_
4 8 12 15
19.4 (2.4) 4.1 (0.5) 148.6 120.0
conditions
0.5 (0.1) 0.2 (0.1) 4.3 3.1
by feeding 24 h per day.
previously fed the K and KM diets exhibited extremely high SGRs when fed the AS diet (4.5-4.0 and 4.3-3.1% day-‘, respectively). In contrast, animals fed the AS diet throughout did not grow as rapidly during this period (3.3-2.1% day- I), suggesting the presence of compensatory growth in these organisms. Nevertheless, these differences were not statistically significant across diets (F2 2 = 13.0, P < 0.05) or between times (F,., = 16.2, P < 0.05) (two-way ANOVA test v&out replication).
0.6 -
3 E
l
Abalone
. .
Kelp meal diet (KM)
silage diet (AS)
Fresh kelp(K)
0.6 -
.P f *
0.4 -
m” 0.2 -
0.0
’,
,
,
,
,
,
r
,
0
2
4
6
6
10
12
14
Time (weeks) Fig. 4. Experiment 2. Body weight (g) of abalone fed with artificial diets containing heated abalone viscera silage (AS), kelp meal (KM) and a natural diet of fresh kelp (K). Means and standard errors are given. Arrow indicates a shift to AS diet.
96
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140 (1996) 87-98
4. Discussion
Artificial diets containing fish silages as protein sources produced superior growth rates of H. jUgens compared with natural macroalgae. No significant differences in growth responses were observed between abalone fed heated (average 53 p,rn day-’ > and unheated (average 6 1 p,rn day- I ) fish silage diets, suggesting that they can utilize the protein from these silages with similar efficiency. Nevertheless, diets containing heated silage had a lower soluble protein content and free amino acid content compared with the unheated silage diet. These results suggest that abalone may be able to utilize free amino acids, or that 32-33% protein in the diet is higher than is required for abalone. No enzyme activity was detected in the pellets at pH 5, and therefore it cannot be assumed that the enzymes in the silage play a role in enhancing protein digestibility in abalone. No significant differences in protein leaching occurred between heated and unheated diets at 20 or 24°C but the unheated silage-based diets lost significantly more protein at 16°C although this diet was more stable in terms of dry matter. Thus, it appears that protein leaching is independent of dry matter loss in water. Clearly, more experiments are need to determine the role of hydrolysis in controlling nutrient availability and solubility. The artificial diet containing abalone viscera as a protein source produced higher growth rates (average 50 pm day-’ ) than the artificial diet based on kelp meal (average 18 km day-‘) or fresh kelp (average 12 km day-‘). The lower growth rates of all abalone during the first phase of the experiment may be due to shorter feed exposure and/or the greater stress the animals were exposed to during this period. It has been reported that abalone are easily stressed by handling and light (Hahn, 1989). No correlation was found between external variables (temperature, oxygen and ammonia levels) and growth, suggesting that stress was the cause of the different growth performances over time. These results suggest that the dietary value of the silage-based diets would be far greater than reported here if animals were not handled so frequently. The results also suggest that the average growth rate of 122 p_rnday-’ produced by the less stressed animals feeding on the abalone diet containing abalone viscera silage more closely reflects dietary value. Growth of abalone on diets of kelp meal and fresh kelp were significantly lower than those on the diet containing abalone viscera silage. Previous reports have shown that algal diets of single species do not provide a nutritionally balanced diet (Day and Fleming, 1992; Viana et al., 1993a). In nature abalone eat many species of macro- and microalgae, which together presumably supply a balanced diet. An artificial diet based on 82% kelp meal appears to have a higher nutritional value than fresh kelp alone. Not only did abalone grow marginally faster on this diet, but they grew faster when switched onto the abalone viscera diet. This may be because the artificial diet of kelp meal offered a more nutritionally balanced diet than fresh kelp alone. Alternatively, it may be due to a higher consumption rate on the artificial diet, a higher concentration of nutrients or a nutritional degradation of the fresh kelp owing to decomposition while in the tanks. Interestingly, the growth of animals feeding on the diet containing abalone viscera after previously feeding on the kelp meal diet was higher than that of animals fed the abalone viscera diet throughout. It has been reported that an animal temporarily deprived
M.T. Viana et al./Aquaculture
140 (1996) 87-98
97
of a nutritionally balanced diet will grow more rapidly when the diet improves to make up for the loss (Lovell, 1994). In this study, the nutritionally deficient abalone were able to compensate by growing rapidly as soon as they were switched to the diet based on abalone viscera silage. Fish and abalone viscera silage are suitable, inexpensive protein sources for abalone diets. The use of abalone viscera is potentially a profitable solution to a waste problem of the abalone fishery. The degree of hydrolysis when protein is balanced to 30% seems not to influence the availability of amino acids when fed to abalone.
Acknowledgements
We would like to thank Francisco Fonseca and the Cooperative of Bahia Tortugas for the contribution of animals and Dr. Scoresby Shepherd for his comments, and especially Dr. Ann Fleming for helpful criticism and editorial comments. This study was supported by a grant from CONACYT No. A9107-0237.
References AOAC, 1990. Offtcial Methods of Analysis, 15th edn. Association of Official Analytical Chemists, Washington, DC, 1298 pp. Austreng, E., 1982. Ensilece fiskeavfall i tiskeoppdmtt. In: Husdyrforsciksmotet. Aktuelt fra statens fagtjeneste for landbruket, NLH, As, Norway, Vol. 1, pp. 525-530. Barret, A.J., 1972. Lysosomal enzymes. In: J.T. Dingle (Editor), Lysozymes, a Laboratory Handbook. North-Holland, Amsterdam, pp. 46-135. Bligh, E.G. and Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37(8): 911-917. Coutteau, P. and Sorgeloos, P., 1992. The use of algal substitute and the requirement for live algae in the hatchery and nursery rearing of bivalve mollusc: An international survey. J. Shellfish Res., 11: 467-476. Curatolo, A., Ryan, M.J. and Mercer, J.P., 1993. An evaluation of the performance of manila clam spat (Tapes philippinarum) fed on different rations of spray-dried algae (Tetraselmis suecicu). Aquaculture, 112: 179-186. Day, R.W. and Fleming, A.E., 1992. The determinants and measurements of abalone growth. In: S.A. Shepherd, M.J. Tegner and S.A. Guzm&t de1 Pr60 (Editors), Abalone of the World. Biology, Fisheries and Culture. Fishing News Books, Oxford, pp. 141- 168. Espe, M., 1993. Studies on the utilization of pre-digested fish proteins in Atlantic salmon (S&no s&r). Ph.D. Thesis, University of Bergen and InStiNte of Nutrition Directorate of Fisheries, Bergen. Espe, M., Lied, E. and Torrisen, K.R., 1993. Changes in plasma and muscle free ammo acids in Atlantic salmon (Salvo s&r) during absorption of diets containing different amounts of hydrolysed cod muscle protein. Comp. Biochem. Physiol., 105A(3): 555-562. Gildberg, A., 1982. Autolysis of fish tissue. General aspects. Thesis for the degree of Doctor Scientiamm, Tromso. Gildberg, A., 1988. Aspartic proteinases in fishes and aquatic invertebrates. Comp. B&hem. Physiol., 91B(3): 425-435. Hahn, K., 1989. Nutrition and growth of abalone. In: K. Hahn (Editor), Handbook of Culture of Abalone and other Marine Gastropods. CRC Press, Boca Raton, FL, pp, 135-180. Johnson, W.C. and Lindsay, A.J., 1939. An improved universal buffer. Analyst London, 64: 490-492. Lovell, T., 1994. Compensatory gain in fish. Aquacult. Mag., 20(l): 91-93.
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Lowry, O.U.. Rosebrough, N.J., Farr, A.L. and Randal, P.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem., 193: 265-275. Nie, Z.-Q., Wang, Z.-Q. and Yan, J.-P., 1986. Experiments on preparing formulated feed and feeding efficiency of young abalone, Haliotis discus hmnai Ino. Mar. Fish. Res., 7: 53-64. Raa, J. and Gildberg, A., 1982. Fish silage: A review. CRC Crit. Rev. Food Sci. Nutr., 116: 343-419. Raa, J., Gildberg, A. and Strom, T., 1983. Silage production: Theory and practice. In: D.A. Lcward, A.V. Taylor and R.A. Lawrie (Editors), Upgrading Waste for Feeds and Food. Vol. 8. Butterworths, London, pp. 117-132. Stone, F.E., Hardy, R.W.. Shearer, K.D. and Scott, T.M., 1989. Utilization of fish silage by rainbow trout (&&no gairdneiri). Aquaculture, 76: 109- 118. Uki, N. and Watanabe, T., 1992. Review of the nutritional requirements of abalone (Haliotis spp.) and development of more efficient artificial diets. In: S.A. Shepherd, M.J. Tegner and S.A. Guzm&t de1 P&I (Editors), Abalone of the World. Biology, Fisheries and Culture. Fishing News Books, Oxford, pp. 504-517. Uki, N., Kemuyama, A. and Watanabe, T., 1985. Nutritional evaluation of several protein sources in diets for abalone Haliotis discus hamai. Bull. Jpn. Sot. Sci. Fish., 51: 1835-1839. Viana, M.T., Mpez, L.M. and Salas, A., 1993a. Diet development for juvenile abalone Haliotis fulgens. Evaluation of two artificial diets and macroalgae. Aquaculture, 117: 149- 156. Viana, M.T., Nava, C. and Solana-!&sores, R., 1993b. Acid fish silages. Effect of preheating and addition of phosphoric and citric acids on the biochemical quality. Ciencias Mar., 19f4): 415-433. Zar, J.H., 1984. Biostatistical Analysis. Prentice Hall, Englewood Cliffs, NJ, 718 pp.
Aquaculture 140 (1996) 87-98
The use of silage made from fish and abalone viscera as an ingredient in abalone feed Maria Teresa Viana *, Lus M. L6pez, Zaul Garcia-Esquivel, Elda Mendez Institute de Investigaciones Ocean&gicas,
Universidad Aut&oma de Baja California, PO Box 4.53, 22 800 Ensenada, BC. M&co
Abstract Silage prEpared from fish and abalone viscera are effective dietary protein sources for the juvenile abalone, Hal&is filgens. Significantly higher growth rates occurred when abalone were fed artificial diets containing heated fish silage (53 p,rn day-‘) and unheated fish silage (61 p,m day- ‘) as a protein source compared with the kelp, Mucrocystis pyrifera (1.5 pm day- ‘>. However, no differences were found between diets containing heated and unheated fish silage at 30% protein inclusion, suggesting that the degree of hydrolysis did not affect protein utilization by abalone. Similar results were obtained when abalone viscera silage was used (50 p,m day-‘), producing faster growth rates than kelp (18 pm day-‘) or kelp meal (12 p,m day-‘). In the same experiment a significant increase in growth rate was observed when abalone with low growth rates, resulting from feeding on kelp and kelp meal, were switched to a diet containing abalone viscera silage. These animals exhibited higher growth rates (135 and 167 km day-‘) than animals fed this diet throughout the trial (122 p,rn day-‘), suggesting the presence of compensatory growth of organisms. Keywords: Abalone; Artificial diets; Diets; Feed; Growth; Protein
1. Introduction Artificial diets important as an Sorgeloos, 1992; plates containing organisms reach
based on less expensive protein sources are becoming increasingly alternative to live feeds in the aquaculture industry (Coutteau and Curatolo et al., 1993). Recently set abalone spat are usually reared on diatom films as feed and weaned to fresh macroalgal diets when the a size of about 3-4 mm (Hahn, 1989). Nevertheless, it has been
* Corresponding author. 00448486/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0044-8486(95)01 196-X
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documented that H. discus, H. discus hannai and H. sieboldii fed with various artificial diets grow faster than those fed with natural algae (Nie et al., 1986; Hahn, 1989). The faster growth rates were attributed to the higher protein content and nutritional quality of the feed. Research conducted by Uki et al. (1985) showed that abalone growth rate was faster when fed a diet supplemented with casein compared with other protein sources. Clearly, casein was used more efficiently by abalone. Thus, inexpensive, high quality protein is an essential element for profitability in the aquaculture industry. Siiage has proved to be a good protein source for sea-farmed fish (Raa and Gildberg, 1982). Fish silage preserved by adding acid (a combination of organic and inorganic acids) is acceptable to salmonids and the nutritional value is as good as that of the fresh, raw material (Austreng, 1982). However, the preservation process causes fish silage to become liquefied, and hence large volumes of dry binder meals are needed to produce a moist pellet of acceptable consistency, resulting in formulation problems. In such a moist feed, 60-70% of the dry matter may result from the added dry meal (Raa and Gildberg, 1982; Raa et al., 1983). During prolonged storage, large amounts of free amino acids may be present in silages owing to the activity of endogenous enzymes in fish tissues. These enzymes are mostly proteases, such as pepsin-type stomach proteases, and lysosomal enzymes, like cathepsin D, which is the major muscle protease (Gildberg, 1982; Gildberg, 1988). Extensive hydrolysis may result in a reduction of the nutritional value of silage. When mixtures of free amino acids are fed, only the absorption mechanism for free amino acids is utilized. As a result, more of the ingested amino acids may be lost in the faeces due to increased competition between amino acid carriers (Espe, 1993). In addition, large amounts of free amino acids present in the pellets may augment protein leaching. It has been shown that partially hydrolysed material is superior in nutritional value to fully hydrolysed material (Stone et al., 1989; Espe et al., 1993). In order to avoid excessive hydrolysis during prolonged storage, enzyme activity from the proteases present in fish silage can be inactivated by heating the raw fish material for 5 min at 60°C prior to ensilaging (Viana et al., 1993b). This results in a 15% reduction in free amino acid levels after 90 days compared with unheated silage. The use of fish and abalone viscera silage as alternative protein sources for abalone could result in cheaper artificial feeds, making their culture more economically viable. The aim of the present study was to test whether diets made with abalone viscera silage and heated and unheated fish silage from mackerel (Scomber juponicus) had a significant effect on the growth rates of the juvenile abalone, H. fulgens. 2. Material and methods 2.1. Diet preparation Silage was made as described by Viana et al. (1993b). In summary, heated (at 60°C for 5 min) and unheated mackerel, S. juponicus (Experiment 1) and heated (at 60°C for 5 min) abalone viscera (Experiment 2) were mixed, when cold, with 2.6% phosphoric acid, 2.6% citric acid and 0.1% sodium benzoate as a preservative. The mixtures were blended to obtain a homogenate and left for 60 days in plastic buckets.
M.T. Viana et al./Aquaculture Table 1 Percentage
composition
Ingredients
of the four artificial Experiment Heated fish silage diet
Silage a Fish meal b Soy bean meal ’ Corn meal d Starch (rice) Kelp meal e Sodium alginate ’ Gelatin (50 blooms) Vitamin mixture g Mineral mixture s Cellulose Methionine
140 (1996) 87-98
89
diets tested, given as a percentage
1
Experiment
of dry matter 2
Abalone viscera silage diet (AS)
Kelp meal diet (KM)
(HS)
Unheated fish silage diet (US)
31.8 0 5 15 10 10 10 6 2 5 5 0.2
31.8 0 5 15 10 10 10 6 2 5 5 0.2
20 16.8 10 10 10 10 10 6 2 5 0 0.2
0 0 0 0 0 82 10 6 2 0 0 0
a Mackerel were kindly supplied by Tecmar, SA, and abalone viscera by the fishery cooperative Bahia Tortugas, BCS.b Supplied by Productos de Ensenada, SA.C 50% protein, kindly supplied by the American Soybean Association.d Mazeca, produced in Mexico? Kindly supplied by CRIP (Centro Regional de Investigaciones Pesqueras, Mexico).’ Kelgin MV, kindly supplied by KeIco.g As recommended by Hahn (1989), using Stay-C from Roche.
Silage diet formulations (Table 1) were based on the constituents recommended by Uki et al. (1985) and Uki and Watanabe (1992). Vitamin and mineral mixtures were those recommended by Hahn (1989). All ingredients were mixed until a complete homogenate was obtained. The diet was then rolled flat to a thickness of 2 mm. Pieces 2 cm x 2 cm were cut for Experiment 1, and pieces 1 cm X 1 cm for Experiment 2. They were then frozen at - 20°C until used. The fish silage pellets used in Experiment 1 were tested for their stability in seawater, both in terms of dry matter and protein. To determine dry matter stability, four pellets of each diet were placed in three control buckets and their dry matter loss was measured after 12 h at 20°C. In addition, a control bucket without animals was used for each diet during Experiments 1 and 2 to measured the daily dry matter loss throughout the experiment. Protein leaching was determined at various temperatures by immersing pellets in seawater at constant turbulence and at 16, 20 and 24°C. A 1 ml sample was taken from the remaining material at 2, 4, 6 and 12 h and its soluble protein content was determined using the Lowry method (Lowry et al., 1951). As enzyme activity is expected in the unheated silage (Gildberg, 19821, the activity present in the fish silage diets was tested. Enzymatic activity was measured at pH 3 following the method of Barret (1972). The incubation mixture consisted of 0.5 ml Johnson/Lindsay buffer (Johnson and Lindsay, 19391, 0.25 ml sample extract and 0.25 ml 8% haemoglobin. The concentration of folin-positive material in the supematant, after adding trichloroacetic acid (TCA, 5% end concentration), was determined by
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90
140 (1996) 87-98
Lowry’s method (Lowry et al., 1951) using tyrosine as a standard. Enzyme activity was also measured at pH 5, which is typical of abalone stomach pH (determined from 20 animals collected from the sea). Azocasein was used as a substrate. Azocasein solution (0.25 ml of a 6% solution dissolved in distilled water) was incubated with 0.65 ml citrate-phosphate buffer and 0.1 ml pellet extract. After 40 min at 37”C, the reaction was stopped by the addition of 3 ml of 3% TCA. The mixture was filtered and the amount of trichloroacetic acid-soluble peptides was measured at 360 nm. To assess the dietary value of the artificial diets, a control diet of fresh kelp (K) was used in Experiments 1 and 2 and an artificial diet made from kelp meal (M. pyriferu) (KM) was used in Experiment 2. Pellets were made by adding 27% water to a mixture of gelatin, sodium alginate, vitamins and dried kelp meal (Table 1). 2.2. Proximate analysis Percentage dry weight was calculated as the dried residue weight of triplicate samples (4-5 g> of each diet after being dried to constant weight at 100°C. Total nitrogen was determined using triplicate samples analyzed by the Kjeldahl method (AOAC, 1990). Crude protein was calculated as %N X 6.25. Total lipid was determined using triplicate samples (10 g) by a column procedure utilizing methanol-chloroform/water as the eluting solvent (Bligh and Dyer, 1959). Ash was determined by heating duplicate samples (3 g) of each diet to 600°C for 18 h. 2.3. Experimental procedure 2.3.1. Experiment 1 Six-month-old H. $&ens with an average shell length of 18.3 mm (SE 0.66) and average weight of 0.6 g (SE 0.06) were obtained from the laboratory of Bahia Tortugas BCS. The abalone were held in flow-through, aerated seawater with a controlled flow of 300 ml min- ‘. Daily average temperatures ranged from 20 to 23°C throughout the feeding trial. Oxygen, pH and ammonium content were monitored (data not shown). Feeding experiments were conducted after 21 days of acclimation to laboratory conditions. During this period abalone were fed fresh M. pyriferu. The effects on growth rate of the artificial diets containing unheated (raw) mackerel silage (US) and heated mackerel silage (HS) were compared with those on a control diet of the fresh kelp, M. pyriyeru (K). Animals were held in 20 1 plastic containers (three replicates per treatment) with 32 abalone per container. Animals were marked with plastic tags so that individual growth could be monitored. Diets were offered for 12 h overnight (ad libitum) every night for 25 weeks. Any remaining food was collected daily. An attempt was made to estimate the food conversion efficiency, but the results were unreliable because of inaccurate recovery of the uneaten pellets. Twice a week algae growing on the inside walls of the containers were removed with a soft brush. Whole-body weight was measured monthly with an electronic balance (0.001 error) and shell length with electronic digital calipers (fO.O1 pm). Specific growth rate (SGR, % day- ’ > was estimated according to Espe (1993): SGR = lOO{(In final weight - In start weight) /days of experiment}
M.T. Viatm et aL/Aquacolture
140 (1996) 87-98
91
2.3.2. Experiment 2 Two-month-old abalone with an average shell length of 8.9 mm (SE 0.93) and weight of 0.1 g (SE 0.01) were obtained from the laboratory of Bahia Tortugas BCS and held in the same conditions as in Experiment 1. Feeding experiments were conducted using four replicate groups of 32 abalone for each diet treatment. Animals were acclimatised to laboratory conditions for 35 days and fed an artificial diet containing abalone viscera silage. Daily average temperature varied from 21 to 24°C throughout the experimental trial. During the experiment, abalone were fed ad libitum for 12 h overnight for 8 weeks. One group was fed the fresh kelp, M. pyrijeru (K), the second was offered the kelp meal diet (KM) and the third the artificial diet containing heated abalone viscera silage (AS) as a protein source. After the 8th week all animals were transferred to the AS diet for an additional 7 weeks. During this time, no container replication was used and food was offered to satiation 24 h per day. Twice a week, all algae growing on the inside walls of the containers were removed with a soft brush. Whole-body weight was measured monthly with an electronic balance (0.001 error) and shell length with digital calipers. 2.4. Statistical
analyses
In Experiment 1, a one-way ANOVA or a two-sample t-test (Zar, 1984) was used at specific times in order to compare mean specific growth rates (SGR) across diet treatments. A one-way repeated ANOVA compared SGR over time, followed by a multiple comparison of the means (SNK, Student-Newman-Keuls test). Shell length was compared over time with a repeated ANOVA test, while comparison across diet treatments was performed with a Kruskal-Wallis range test, followed by non-parametric Dunn’s multiple comparisons (Zar, 1984) owing to violations of homoscedasticity in the raw data. A two-way ANOVA test compared SGR of abalone across diet and time treatments during the first 8 weeks of Experiment 2 (3 diets X 2 times X 4 replicates). The ANOVA test was followed by multiple comparisons of the means (SNK test) to identify the groups which differed. Since no replicate containers were used in the second half of the experiment, a two-way ANOVA test without replication was used to compare SGR between diets (3 diets X 2 times). The computer package of Sigma-Stat for DOS was used in these statistical analyses.
3. Results 3.1. Experiment
I
The fish silage diets were similar in protein (31-33%) and lipid (3-5%) while the fresh kelp diet was lower in both protein (2%) and lipid (0.1%) (Table pellet stability test indicated a dry matter loss of 10.0% (SE 0.4) for the HS 2.6% (SE 0.3) for the US diet after 12 h submersion at 20°C. Significantly (P < 0.05) mean protein leaching occurred at 20 and 24°C compared with 16°C
content, 2). The diet and greater for both
M.T. Viana et al./Aquaculture
92 Table 2 Proximate
analysis
of the diets used in Experiments
140 (1996) 87-98
1 and 2, expresed as a percentage
Diet
Crude protein
Total lipid
Ash
Water
Heated fish silage (HS) Unheated fish silage (US) Abalone viscera silage (AS) Kelp meal (KM) Fresh kelp (K)
33.1 31.9 31.2 5.1 2.0
3.8 3.3 4.7 0.3 0.1
18.2 16.1 18.9 14.1 12.0
24.1 29.6 46.6 31.0 84.0
the US and HS diets. No significant differences (P > 0.05) occurred between the US and HS diets at 20 and 24°C. However, the US diet incubated at 16°C exhibited significantly greater (P < 0.05) mean protein leaching than the HS diet (98.7 vs. 90.1 mg g-l protein, respectively). At pH 3, the HS diet showed a remaining enzyme activity of 0.36 krnol tyr eq ml-’ h- ’ compared with 2.15 for the US diet. No activity was registered when pellets were assayed at pH 5. Abalone fed silage diets exhibited significantly different (P < 0.05) shell growth rates over time (F’s = 55.1 and F,*s = 45.2 for HS and US diets, respectively) (Fig. 1, Table 3). During the first month abalone growth was slow (3.0 and 2.0 pm day-’ on the HS and US diets, respectively), but gradually increased to 46-65 pm day- ’ (average 53 km day-’ ) on the HS diet and between 52 and 67 pm day-’ (average 61 p,rn day-‘) on the US diet. Animals fed the control diet K grew at significantly lower (P < 0.05) growth rates during the first 2 months (l-2 pm day-‘), and subsequently died (Fig. 1, Table 3). Significantly different shell growth rates (Kruskal-Wallis test, H = 30.15, df = 2, P < 0.001) occurred between diet treatments after the second month (end of kelp treatment), but no differences were detected between animals fed silage-based diets at
30 26 26E g
24-
r_ p
22-
u = a,
20 -
l
’ .
Heated silage diet (HS) Unheated
silage diet (US)
Fresh kelp (K)
??!I 16 -
0
5
10
15
20
25
30
Time (weeks) Fig. 1. Experiment 1. Shell length (mm) of abalone fed with heated (HS) and unheated and fresh kelp (K). Means and standard errors are given.
(US) mackerel
silage
M.T. Viana et al,/Aquacubure
I40 (1996) 87-98
93
Table 3 Growth rates of juvenile abalone fed for 7 months on artificial diets containing heated mackerel unheated silage (US) and a natural diet of fresh kelp (K). Standard errors in parentheses Diet
No. of weeks
HS
0 4 8 13 17 22 25
us
K
silage (HS),
Growth rate (km day-‘)
(% day- ‘)
3.1 34.4 46.2 55.1 47.1 64.2
0.5 (0.1) 0.7 (0.3) 0.9 (0.1) 0.8 (0.1) 0.9 (0.1) l.O(O.1)
(1.2) (3.2) (4.1) (3.1) (2.6) (3.9)
0
4 8 13 17 22 25
1.6 (0.3) 22.3 (3.5) 51.5 (4.1) 63.5 (7.3) 61.5 (7.1) 67.4 (4.7)
0.5 0.7 1.0 0.8 0.9 0.8
0 4 8
1.2(0.5) 2.4 (0.8)
0.2 (0.1) -0.3 (0.1)
(0.1) (0.1) (0.1) (0.1) (0.1) (0.1)
this time (Dunn’s multiple comparison, P < 0.05) or by the end of the experiment (two-sample test, t = 0.523, df = 50, P = 0.603) (Table 3). Body weight gain followed a similar pattern to shell length (Fig. 2). The one-way ANOVA indicated significantly lower SGRs for abalone fed the K diet during the first (F2,63 = 16.3; P < O.OOl>and second (F2v63= 82.1; P < 0.001) month of the experiment compared with those offered silage diets (Table 3). Animals fed the K 2.4
-
2.0 -
l
Heated silage (HS)
l
Unheated
A
Fresh kelp (K)
silage (US)
s E .P
1.6 -
g &
1.2 -
$
0
5
10
15
20
25
30
Time (weeks) Fig. 2. Experiment 1. Body weight (g) of abalone fed with heated (HS) and unheated (US) mackerel silage and fresh kelp (K). Means and standard errors are given.
M.T. Viana et al./Aquaculture
94
140 (1996) 87-98
diet exhibited a negative SGR during the second month ( - 0.3% day- ’ >, just prior to their death. The SGRs of animals feeding on the silage diets were low for the first month (0.5% day-’ ), but subsequently maintained a constant SGR of 0.9% day-’ until the end of the trial (Table 3). 3.2. Experiment
2
The mean shell length of abalone was significantly different between diets (F2 = 38.1, P < 0.001) and between times (Fz = 53.6, P < 0.001; Fig. 3). A significant interaction
effect between time and diet was observed during the first half of the experiment (F4 = 12.6, P < C.OOl), owing to the sustained shell growth of animals fed the AS diet (average 50 pm day-’ > in contrast to the halt in growth of animals feeding on the KM and K algal diets (Fig. 3). A change in shell pigmentation was associated with the diets. The AS diet produced an intense blue-coloured shell, while the KM and K diets produced a light blue shell. Once all animals were switched to the AS diet, growth rates in terms of length increased markedly (Table 4). The increase in growth rate at this time (average 122 pm day-‘) by the animals fed the AS diet throughout suggests that changes in the experimental procedure influenced the results. Nevertheless, at the end of the experiment animals previously fed the KM diet were similar in length to those fed the AS diet throughout (Fig. 3). Body weight gain followed a similar pattern to shell length (Fig. 4). SGRs decreased during the first half of the experiment and subsequently recovered during the last 8 weeks (Table 4). A two-way ANOVA test indicated highly significant effects of diet (Fz,,8 = 104.6, P < 0.001) and time factors (F,,,s = 169.4, P < 0.001) during the first half of the experiment, with higher values observed for animals fed the AS diet, followed by those fed KM and K (Table 4). A significant interaction term between diet and time treatments was also found (F2.t8 = 8.2, P < 0.003). Both groups
20 16 -
E
16-
z. 5
14-
@ 3 z
12 -
l
Abalone silage diet (AS)
.
Kelp meal diet (KM)
A
Fresh kelp(K)
5 10 -
0I
I
I
I
I
I
I
I
0
2
4
6
6
10
12
14
Time (weeks) Fig. 3. Experiment 2. Shell length (mm) of abalone fed with artificial diets containing heated abalone viscera silage (AS), kelp meal (KM) and a natural diet of fresh kelp (K). Means and standard errors are given. Arrow indicates a shift to AS diet.
M.T. Viana et al./Aqunculture
I40 (1996) 87-98
95
Table 4 Growth rates of juvenile abalone fed for 8 weeks on artificial diets containing abalone viscera silage (AS), kelp meal (KM) and a natural diet of fresh kelp (K). The KM and K treatments were switched to the AS diet after the 8th week. Standard errors in parentheses Diet
AS
KM
Diet change +
K
Diet change --t
’ Change of experimental
No. of weeks
Growth rate
(km day- ’ )
(% day- ‘)
0 4 8 12 a 15
64.9 (1.8) 34.6 ( 14.7) 132.9 a 111.4
2.2 (0.1) 1.4(0.1) 3.3 a 2.1
0 4 8 12 15
30.4C1.5) 5.6 (4.1) 155.7 177.1
1.1 (0.1) 0.1 (0.1) 4.5 4.0
0
_
4 8 12 15
19.4 (2.4) 4.1 (0.5) 148.6 120.0
conditions
0.5 (0.1) 0.2 (0.1) 4.3 3.1
by feeding 24 h per day.
previously fed the K and KM diets exhibited extremely high SGRs when fed the AS diet (4.5-4.0 and 4.3-3.1% day-‘, respectively). In contrast, animals fed the AS diet throughout did not grow as rapidly during this period (3.3-2.1% day- I), suggesting the presence of compensatory growth in these organisms. Nevertheless, these differences were not statistically significant across diets (F2 2 = 13.0, P < 0.05) or between times (F,., = 16.2, P < 0.05) (two-way ANOVA test v&out replication).
0.6 -
3 E
l
Abalone
. .
Kelp meal diet (KM)
silage diet (AS)
Fresh kelp(K)
0.6 -
.P f *
0.4 -
m” 0.2 -
0.0
’,
,
,
,
,
,
r
,
0
2
4
6
6
10
12
14
Time (weeks) Fig. 4. Experiment 2. Body weight (g) of abalone fed with artificial diets containing heated abalone viscera silage (AS), kelp meal (KM) and a natural diet of fresh kelp (K). Means and standard errors are given. Arrow indicates a shift to AS diet.
96
M.T. Viam et nL/Aquacubure
140 (1996) 87-98
4. Discussion
Artificial diets containing fish silages as protein sources produced superior growth rates of H. jUgens compared with natural macroalgae. No significant differences in growth responses were observed between abalone fed heated (average 53 p,rn day-’ > and unheated (average 6 1 p,rn day- I ) fish silage diets, suggesting that they can utilize the protein from these silages with similar efficiency. Nevertheless, diets containing heated silage had a lower soluble protein content and free amino acid content compared with the unheated silage diet. These results suggest that abalone may be able to utilize free amino acids, or that 32-33% protein in the diet is higher than is required for abalone. No enzyme activity was detected in the pellets at pH 5, and therefore it cannot be assumed that the enzymes in the silage play a role in enhancing protein digestibility in abalone. No significant differences in protein leaching occurred between heated and unheated diets at 20 or 24°C but the unheated silage-based diets lost significantly more protein at 16°C although this diet was more stable in terms of dry matter. Thus, it appears that protein leaching is independent of dry matter loss in water. Clearly, more experiments are need to determine the role of hydrolysis in controlling nutrient availability and solubility. The artificial diet containing abalone viscera as a protein source produced higher growth rates (average 50 pm day-’ ) than the artificial diet based on kelp meal (average 18 km day-‘) or fresh kelp (average 12 km day-‘). The lower growth rates of all abalone during the first phase of the experiment may be due to shorter feed exposure and/or the greater stress the animals were exposed to during this period. It has been reported that abalone are easily stressed by handling and light (Hahn, 1989). No correlation was found between external variables (temperature, oxygen and ammonia levels) and growth, suggesting that stress was the cause of the different growth performances over time. These results suggest that the dietary value of the silage-based diets would be far greater than reported here if animals were not handled so frequently. The results also suggest that the average growth rate of 122 p_rnday-’ produced by the less stressed animals feeding on the abalone diet containing abalone viscera silage more closely reflects dietary value. Growth of abalone on diets of kelp meal and fresh kelp were significantly lower than those on the diet containing abalone viscera silage. Previous reports have shown that algal diets of single species do not provide a nutritionally balanced diet (Day and Fleming, 1992; Viana et al., 1993a). In nature abalone eat many species of macro- and microalgae, which together presumably supply a balanced diet. An artificial diet based on 82% kelp meal appears to have a higher nutritional value than fresh kelp alone. Not only did abalone grow marginally faster on this diet, but they grew faster when switched onto the abalone viscera diet. This may be because the artificial diet of kelp meal offered a more nutritionally balanced diet than fresh kelp alone. Alternatively, it may be due to a higher consumption rate on the artificial diet, a higher concentration of nutrients or a nutritional degradation of the fresh kelp owing to decomposition while in the tanks. Interestingly, the growth of animals feeding on the diet containing abalone viscera after previously feeding on the kelp meal diet was higher than that of animals fed the abalone viscera diet throughout. It has been reported that an animal temporarily deprived
M.T. Viana et al./Aquaculture
140 (1996) 87-98
97
of a nutritionally balanced diet will grow more rapidly when the diet improves to make up for the loss (Lovell, 1994). In this study, the nutritionally deficient abalone were able to compensate by growing rapidly as soon as they were switched to the diet based on abalone viscera silage. Fish and abalone viscera silage are suitable, inexpensive protein sources for abalone diets. The use of abalone viscera is potentially a profitable solution to a waste problem of the abalone fishery. The degree of hydrolysis when protein is balanced to 30% seems not to influence the availability of amino acids when fed to abalone.
Acknowledgements
We would like to thank Francisco Fonseca and the Cooperative of Bahia Tortugas for the contribution of animals and Dr. Scoresby Shepherd for his comments, and especially Dr. Ann Fleming for helpful criticism and editorial comments. This study was supported by a grant from CONACYT No. A9107-0237.
References AOAC, 1990. Offtcial Methods of Analysis, 15th edn. Association of Official Analytical Chemists, Washington, DC, 1298 pp. Austreng, E., 1982. Ensilece fiskeavfall i tiskeoppdmtt. In: Husdyrforsciksmotet. Aktuelt fra statens fagtjeneste for landbruket, NLH, As, Norway, Vol. 1, pp. 525-530. Barret, A.J., 1972. Lysosomal enzymes. In: J.T. Dingle (Editor), Lysozymes, a Laboratory Handbook. North-Holland, Amsterdam, pp. 46-135. Bligh, E.G. and Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37(8): 911-917. Coutteau, P. and Sorgeloos, P., 1992. The use of algal substitute and the requirement for live algae in the hatchery and nursery rearing of bivalve mollusc: An international survey. J. Shellfish Res., 11: 467-476. Curatolo, A., Ryan, M.J. and Mercer, J.P., 1993. An evaluation of the performance of manila clam spat (Tapes philippinarum) fed on different rations of spray-dried algae (Tetraselmis suecicu). Aquaculture, 112: 179-186. Day, R.W. and Fleming, A.E., 1992. The determinants and measurements of abalone growth. In: S.A. Shepherd, M.J. Tegner and S.A. Guzm&t de1 Pr60 (Editors), Abalone of the World. Biology, Fisheries and Culture. Fishing News Books, Oxford, pp. 141- 168. Espe, M., 1993. Studies on the utilization of pre-digested fish proteins in Atlantic salmon (S&no s&r). Ph.D. Thesis, University of Bergen and InStiNte of Nutrition Directorate of Fisheries, Bergen. Espe, M., Lied, E. and Torrisen, K.R., 1993. Changes in plasma and muscle free ammo acids in Atlantic salmon (Salvo s&r) during absorption of diets containing different amounts of hydrolysed cod muscle protein. Comp. Biochem. Physiol., 105A(3): 555-562. Gildberg, A., 1982. Autolysis of fish tissue. General aspects. Thesis for the degree of Doctor Scientiamm, Tromso. Gildberg, A., 1988. Aspartic proteinases in fishes and aquatic invertebrates. Comp. B&hem. Physiol., 91B(3): 425-435. Hahn, K., 1989. Nutrition and growth of abalone. In: K. Hahn (Editor), Handbook of Culture of Abalone and other Marine Gastropods. CRC Press, Boca Raton, FL, pp, 135-180. Johnson, W.C. and Lindsay, A.J., 1939. An improved universal buffer. Analyst London, 64: 490-492. Lovell, T., 1994. Compensatory gain in fish. Aquacult. Mag., 20(l): 91-93.
98
M.T. Viana et al./Aquaculture
140 (1996) 87-98
Lowry, O.U.. Rosebrough, N.J., Farr, A.L. and Randal, P.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem., 193: 265-275. Nie, Z.-Q., Wang, Z.-Q. and Yan, J.-P., 1986. Experiments on preparing formulated feed and feeding efficiency of young abalone, Haliotis discus hmnai Ino. Mar. Fish. Res., 7: 53-64. Raa, J. and Gildberg, A., 1982. Fish silage: A review. CRC Crit. Rev. Food Sci. Nutr., 116: 343-419. Raa, J., Gildberg, A. and Strom, T., 1983. Silage production: Theory and practice. In: D.A. Lcward, A.V. Taylor and R.A. Lawrie (Editors), Upgrading Waste for Feeds and Food. Vol. 8. Butterworths, London, pp. 117-132. Stone, F.E., Hardy, R.W.. Shearer, K.D. and Scott, T.M., 1989. Utilization of fish silage by rainbow trout (&&no gairdneiri). Aquaculture, 76: 109- 118. Uki, N. and Watanabe, T., 1992. Review of the nutritional requirements of abalone (Haliotis spp.) and development of more efficient artificial diets. In: S.A. Shepherd, M.J. Tegner and S.A. Guzm&t de1 P&I (Editors), Abalone of the World. Biology, Fisheries and Culture. Fishing News Books, Oxford, pp. 504-517. Uki, N., Kemuyama, A. and Watanabe, T., 1985. Nutritional evaluation of several protein sources in diets for abalone Haliotis discus hamai. Bull. Jpn. Sot. Sci. Fish., 51: 1835-1839. Viana, M.T., Mpez, L.M. and Salas, A., 1993a. Diet development for juvenile abalone Haliotis fulgens. Evaluation of two artificial diets and macroalgae. Aquaculture, 117: 149- 156. Viana, M.T., Nava, C. and Solana-!&sores, R., 1993b. Acid fish silages. Effect of preheating and addition of phosphoric and citric acids on the biochemical quality. Ciencias Mar., 19f4): 415-433. Zar, J.H., 1984. Biostatistical Analysis. Prentice Hall, Englewood Cliffs, NJ, 718 pp.