The effects of semi-purified diets on growth and condition of Sepia officinalis L. (Mollusca: Cephalopoda)

Camp. 3i~~ll~*~l.Pit?_~iol. Vol. 109A. No. 4. pp. lOO7-1016, 1994 Copyright t‘ 1994 Elseviet Science Ltd Printed in Great Britain. All rights reserved 0300-9629/94 67.00 + 0.00

Pergamon 0300-%29(94)00102-2

The effects of semi-purified diets on growth and condition of Sepia oficinalis L. (Mollusca: Cephalopoda) Bernardino

G. Castro* and Phillip G. Lee-f

*Institute de Investigaciones Marinas de Vigo (CSIC), (C) Eduardo Cabello 6, 36208, Vigo (Pontevedra), Spain; and tThe Marine Biomedical Institute, The University of Texas Medical Branch at Galveston, Galveston, TX 77555, U.S.A.

The effects of five different surimi diets (fish myofibrillar protein concentrate) on growth and condition of Sepia oficinalis were evaluated in terms of individual growth using different morphometric and biochemical parameters. A casein supplemented surimi diet produced significant growth (instantaneous growth rate 0.4-0.8% body weight). The digestive glandto-body weight ratio increased and the cuttle~ne-to-body weight ratio decreased significantly in relation to instantaneous growth rate. The RNA content of mantle muscle increased significantly, while the DNA content of mantle muscle did not change in relation to instantaneous growth rate. Mantle muscle protein content was depleted in cuttlefish with instantaneous growth rate ~0. No compensatory food consumption, food conversion or growth was observed for cuttlefish fed surimi diets after refeeding them with a natural diet (thawed raw shrimp). Digestive gland and cuttle~ne-to-~dy weight ratios, and RNA content in mantle muscle could be used as short-term indicators of instantaneous growth rate and condition of cuttlefish. Mantle muscle protein content could be used as a long-term indicator. Key words: Cuttlefish; Nutrition; Semi-purified diets; Surimi; Growth indices; Condition indices; ADN, ARN; Cephalopod culture. Comp. Biochem. Physiol. 109A, 1007-1016, 1994.

Introduction Cephalopods show high metabolic rates and fast growth, which are sustained by high feeding rates (O’Dor and Wells, 1987). Availability of food, together with temperature, is the main external modulator of cephalopod growth. Temperature should affect all individuals of a population more homogeneously than either the availability Correspondence to: B. G. Castro, Instituto de Investigaciones Marinas de Vigo (CSIC), (C) Eduardo Cabello 6, 36208, Vigo (Pontevedra), Spain. Fax 3486292762. Received 6 January 1994; accepted 10 June 1994.

or nutritional quality of food. Therefore, an important part of the individual growth variability within a cohort might be the result of the quality and quantity of food available, but these variables are difficult to evaluate either individually or for a whole population. Individual growth rates of a wild population are not easily measured in cephalopods because their ages usually cannot be determined without wide bias (Voss, 1983). Similar problems have been found in other taxonomic groups (Summerfelt and Hall, 1987) and different indices of growth and condition have been defined with the

1007

1008

B. G. Castro and P. G. Lee

aim of evaluating how different factors affect individual growth and for estimating this growth. These indices are based on different morphometric and biochemical parameters. Morphometric indices have been used for establishing condition in cephalopods, but their validation has rarely been done (Najai, 1984; Castro et al., 1991, 1992). Biochemical indices have been shown to be good indicators of growth and condition in many taxonomic groups but they have been used infrequently for cephalopods (Packard and Albergoni, 1970; Clarke et al., 1989). Both types of indices useful in estimating could be very cephalopod growth and condition. Cephalopods offer a unique model for studying metabolism due to the predominance of their amino acid metabolism (Lee, 1994). Cephalopod body composition ranges between 75 and 85% protein on a dry weight basis. Since cephalopods grow at rapid rates for most of their life cycle, the demands on protein synthesis are also high. Furthermore, the protein/energy required for optimum growth appears to be significantly greater than for other aquatic invertebrates or fishes (> 50 mg protein MJ energy-’ vs. 25-30 mg protein MJ energy-‘; Lee, 1994) and cephalopods are efficient at assimilating proteins (apparent protein digestibility >80; Lee, 1994). In addition, cephalopods digest and assimilate dietary lipids less efficiently than most carnivores and they appear to use little lipid for energy metabolism (O’Dor et al., 1984). Therefore, protein quality or amino acid balance would be the best measure of nutritional value for a diet for cephalopods, not the ratios of the major nutrients and energy which has been used for terrestrial vertebrate nutrition. The development of a prepared diet for growing cephalopods is an important goal for the culture and nutritional physiology of cephalopods (Boletzky and Hanlon, 1983). Recently, a surimi-derived diet (fish myofibrillar protein concentrate) was developed. It was ingested by the cuttlefish Sepia ojicinalis but did not produce growth. It was postulated that the lack of growth was due to the low nutritional quality of the diet and that the diet could be improved with nutrient supplementation (Castro et al., 1993). For this reason, the current growth

trial was designed to evaluate the growth of S. oficinalis when fed different nutrient supplementations of the surimi diet. The results of this growth trial were evaluated for individual growth through a period of feeding with the semi-purified diets followed by a “recovery” period refeeding with a natural food (thawed raw shrimp, Penaeus setferus). Our experimental hypothesis was: “Purified proteins and highly unsaturated lipids will supplement the amino acid and fatty acid composition of surimi diets, resulting in improved growth rates and condition.” Different morphometric and biochemical parameters were measured and used for indicators of growth and condition in cephalopods.

Materials and Methods Forty-two laboratory cultured cuttlefish Sepia ojicinalis (106 k 17.6 g; mean + SD) were equally distributed among six circular tanks (500 1 volume and 1.6 m diameter) connected to a common filtration system. The complete system was similar to typical systems used for the culture of cephalopods (Yang et al., 1989). Water temperature was but was stable at not controlled, 21 + l.O”C. The cuttlefish were tagged with a small numbered plastic sheet fixed to the upper anterior part of the mantle by a plastic thread (FTF-69 Fingerling Tag; Floy tag, Seattle, WA). Seven cuttlefish were distributed into each tank so that there were no significant differences between tanks (P > 0.05) in either the initial wet weights or mantle lengths of the cuttlefish. Each tank was assigned randomly to receive either a control diet, SH (thawed raw shrimp, P. setiferus), a reference surimi diet (RW) or one of four different supplemented surimi diets. The composition of the reference surimi diet, and the four supplemented surimi diets, is shown in Table 1. All diets were prepared using the method described by Castro et al. (1993) Procedure Cuttlefish were transferred to the 500 1 tanks, and then weaned for 14 days from the laboratory maintenance diet of thawed shrimp (P. setiferus) onto the surimi diets. This procedure consisted of progressively increasing the proportion of the surimi diets

1009

Growth and condition in cuttlefish

at each feeding. During this 1Cday transition period, the same weight of food was given to each diet treatment. After the transition period, cuttlefish were fed for 17 days with one of the surimi diets or the thawed shrimp control (semipurified diet period). Every diet was offered in excess. Finally, all cuttlefish were fed thawed shrimp during the 1l-day recovery period. During each experimentation period, the daily amount of food was partitioned by weight into two feedings: 50% of the food was offered at 8.30 and the other 50% at 16.30. Uneaten food was netted out of the tanks 2 hr after feeding. The weight of the food netted out was multiplied by a correction factor to account for water absorption in order to estimate the change in weight. The wet body weight of the cuttlefish (g SW) was measured initially when they were distributed into the tanks and at the end of transition period, semi-purified diet period and recovery period. Using these data, the following information was determined: (1) individual instantaneous growth rate IGR (%BW day-‘) = [(ln I+‘,-In IV,)/t]*lOO, where In is the natural logaritm, IV, and IV, are the initial and final individual wet weight of the cuttlefish, and t is the time of the measuring period in days; (2) feeding rate FR (%BW day--‘) = [FI/average W,,,]*lOO,where FI is the weight of food ingested and average I$‘(,,

is the average wet weight of the cuttlefish in a group over the time interval (t) measured in days; and (3) food conversion FC (%) = [(IV,- WT,)/FI]* 100, where WT, and WT, are the initial and final total wet weight of the cuttlefish in a tank during the period considered. Morphometric and biochemical measures

Three cuttlefish from each diet treatment were killed at the end of the semi-purified diet period, and another two cuttlefish from each diet treatment were killed at the end of the recovery period. A sample of the ventral mantle muscle was taken for biochemical analysis from each cuttlefish. Also, the cuttlebone and digestive gland were dissected and weighed. The rest of the body was dried at 105°C until constant weight and its water content was determined. The digestive gland-tobody (without this organ) weight (DGI) and cuttlebone-to-body (without cuttlebone) weight (CUT) ratios were calculated as percentages. Moreover, the ventral mantle muscles were used to determine: water content after drying the muscle 24 hr at 105°C; DNA and RNA composition using the procedure of Schmidt-Thannhauser (Munro and Fleck, 1966); and protein content by reaction with BCA after incubating samples with 0.5 N NaOH for 14 hr at 30°C (Smith et al., 1985).

Table 1. Food formulation expressed as the percentage of each ingredient in the five surimi diets. EA-L: surimi diet with egg albumin and no menhaden oil. EA: surimi diet with egg albumin. CA: surimi diet with casein. WE: surimi diet with whole egg. RW: control surimi diet. AIN: American Institute of Nutrition. USB: United States Biochemical

Surimi NaCl Mineral mix (AIN 76) Vitamin mix (AIN 76) Arginine (USB) Glutamate (USB) Leucine (USB) Proline (USB) Egg albumin Casein Whole egg Menhaden oil Cholesterol Lecithin

EA-L

EA

CA

WE

RW

88.0 2.0 2.0 2.0

86.0 2.0 2.0 2.0 -

86.0 2.0 2.0 2.0 -

86.0 2.0 2.0 2.0 -

92.0 2.0 2.0 2.0 0.5 0.5 0.5 0.5 -

6.0

6.0

6.0

-

1.0 0.5 0.5

1.0 0.5 0.5

6.0 1.0 0.5 0.5

B. G. Castro and P. G. Lee

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Co-variation of variables with individual IGR was evaluated by means of the Pearson correlation coefficient (single variables) or Spearman correlation coefficient (ratios of variables) (Sokal and Rohlf, 1981). Statistical comparisons between IGR of each diet group were made using the Ryan’s test (Day and Quinn, 1989).

Results Table 2 presents the feeding rates and food conversions during the period in which the cuttlefish were fed surimi diets (semi-purified diet period) and during the period in which the cuttlefish were fed again on thawed raw shrimp (recovery period). The FRs during the semi-purified diet period were lower than during the recovery period, and there were clear differences in FRs among semi-purified diets. The lowest FR corresponded to the whole egg supplemented surimi (WE) and the highest rate to the unsupplemented surimi (RW). The FCs were directly related to the FRs, except with RW that did not produce growth. During the recovery period, all cuttlefish that had been fed surimi diets increased their FR approaching the FR of the cuttlefish fed thawed raw shrimp (SH). The FCs of the cuttlefish that had been fed the CA and RW surimi diets were improved during the recovery period, being close to the FC of the SH diet. In the cases of EA-L, EA and CA diets, the FC increased with respect to the semi-purified diet period but did not reach the level of the SH diet. Table 3 lists the IGRs of each individual cuttlefish during both the semi-purified diet and recovery periods. No significant growth was obtained with any surimi diet except Table 2. Feeding rate (FR) and food conversion (FC) of cuttlefish fed semi-purified diets (EA-L, EA, CA, WE, RW) or shrimp (SH) during 17 days of a semi-purified diet period and shrimp during an 1Iday recovery period. See Table 1 for abbreviations Semi-purified diet period Diet EA-L EA CA WE RW SH

FR 3.15 3.44 4.18 2.45 5.34 7.85

FC -8.28 0.06 8.61 - 13.50 0.79 34.59

Recovery period FR 5.92 7.52 6.60 4.43 7.06 7.03

FC 26.40 26.49 33.42 26.81 35.61 37.59

with CA. The CA diet produced a growth of 16-25% of the growth on the natural thawed raw shrimp diet (SH). After the recovery period, the majority of cuttlefish increased their IGR but they still remained lower than IGRs of the cuttlefish fed shrimp. Only two cuttlefish (in CA and WE groups) lost weight during the recovery period, while three cuttlefish died (in the EA-L, EA and WE groups). There was a significant negative correlation between IGR and CUT (P < 0.001, Fig. 1). The correlation between IGR and positive and significant DGI was (P < 0.001, Fig. 1). Water content of the muscle ranged from 73.1% to 83.0% and correlated with IGR was negatively (Cor = -0.706, P c 0.001). Total water content of the body ranged from 77.2% to 84.0% and was also negatively correlated with IGR (Cor = -0.795, P < 0.001). Both water contents were highly correlated (Cor = 0.934, P < 0.001). The RNA content in mantle muscle increased significantly with IGR (P < 0.001, Fig. 2) while DNA content did not change with IGR (P > 0.05, Fig. 2). Muscle protein content was higher in growing cuttlefish than in starving ones, but it was not correlated clearly with IGR in cuttlefish with IGR > 0 (Fig. 2). The RNA/DNA and RNA/protein ratios inwith IGR significantly creased (Cor = 0.759, P < 0.001 and Cor = 0.744, P < 0.001, respectively) as expected due to RNA increasing with IGR.

Discussion Cuttlefish grow very fast as do other cephalopods (Forsythe and Van Heukelen, 1987). In this experiment, IGR was >2.3% for cuttlefish fed raw shrimp, while only the casein supplemented surimi diet (CA) produced significant growth. Five of the seven cuttlefish fed this diet grew with a rate over 0.4% and one of them reached 0.8% (Table 3). This result confirms our former expectations about the potential of surimi as a diet for cuttlefish (Castro et al., 1993). The unsupplemented surimi diet (reference surimi diet in this experiment, RW) did not produce significant growth (Castro et al., 1993 and the present results). The same happened with the other three protein supplemented surimi diets, two of them similar

1011

Growth and condition in cuttlefish

to the casein supplemented surimi diet except in the kind of protein added (see Table 1). Hence, the addition of casein made the difference. Cephalopods have

metabolic requirements for high-quality protein (Lee, 1994), but specific requirements for amino acids are unknown. It could be that casein satisfies the cuttlefish’s

Table 3. Individual instantaneous growth rates (IGR) of cuttlefish fed different diets during 17 days of a semi-purified diet period and shrimp during an 1l-day recovery period. See Table 1 for diet abbreviations (s: individuals sacrified at the end of one of these periods; d: individuals died during the recovery period. X + SD: mean + standard deviation). Letters after means indicate the statistical significance of differences between IGR for each diet belonging to the same period. Means with the same letter are not significantly different (P > 0.05) Diet EA-L EA-L EA-L EA-L EA-L EA-L EA-L XfSD EA EA EA EA EA EA EA XkSD CA CA CA CA CA CA CA X&SD WE WE WE WE WE WE WE X*SD RW RW RW z RW RW XfSD SH

Individual

number S

S : S S -

8 9 10 11 ,12 13 14 1.5 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

S S i S

S -

S S S S S -

S S : S S S S S

S S -

36

S

S

::: SH ::: SH X&SD

:8’ 39 40 41 42

S S S -

Semi-purified period IGR 0.11 -0.32 -0.54 0.00 0.05 -0.38 -0.54 -0.23 + 0.28 a 0.13 -0.17 0.02 -0.06 0.05 -0.29 -0.03 -0.05 + 0.14 ab 0.47 -0.31 0.40 -0.15 0.81 0.56 0.54 0.33 f 0.41 b -0.60 -0.44 -0.28 -0.01 0.00 -0.41 -0.09 - 0.26 f 0.23 a 0.04 -0.12 0.27 -0.11 -0.08 0.21 0.11 0.05 + 0.16 ab 3.31 2.87 2.53 2.77 2.61 2.85 2.50 2.78 f 0.28 c

Recovery period IGR 776 0.82 1.87 1.48 f 0.58 a 1.13 2.19 2.64 1.99 f 0.78 a -;71 3.00 2.82 2.62 1.93 + 1.77 a -0.35 1.44 2.17 1.09 * 1.30 a 2.18 2.46 2.53 2.97 2.54 + 0.33 a 2.33 2.64 3.04 2.78 2.70 + 0.30 a

IO12

9. G. Castro and P. G. Lee

.

. 0 @O 6-

..

’

.*

2

.

.

‘0

.

l

..

@

0

8

0

COR= 0.894

COR= -0.903

-1

0

2

1

3

4

IGR Fig. 1. Variation of digestive gland to body weight (DGI) and cuttlebone to body weight (CUT) ratios, both expressed as a percentage, with instantaneous growth rate (IGR) of cuttlefish. COR: correlation coefficient between variables. Points inside circles correspond to cuttlefish with significant growth after refeeding with thawed raw shrimp.

amino acid requirements better than egg albumin or whole egg. Table 4 shows the amino acid composition of the supplemented proteins (NRC, 1982; Lentner, 1981) and the mean composition of the mantle muscle of ten cephalopods (Suyama and Kobayashi, 1980; Jhaveri et al., 1984; Iwasaki and Harada, 1985). Considering the relationship between the pattern of essential amino acids in diet and in muscle as a good a priori indicator of the nutritive value of a dietary protein (Cowey and Tacon, 1983), casein and whole egg proteins would have a higher nutritive value for cephalopods than albumin. Correlation be-

tween essential amino acid composition of mantle muscle and that of casein and whole egg is 0.807 in both cases, while it is 0.702 for albumin. The lower growth with whole egg protein could be due to the low FR of WE. Future research must evaluate these results over a longer period and must study specifically the amino acid requirements of cuttlefish. No increase in food consumption, FC or IGR for cuttlefish fed surimi diets was found when compared with the same cuttlefish fed raw shrimp during the recovery period (see Tables 2 and 3). Therefore, no compensatory behavioural or physiological

Growth and condition in cuttlefish

mechanisms seem to exist in cuttlefish, at least in laboratory conditions, for adjusting growth to a fixed rate. As a consequence, the wide size variation among wild cuttlefish of similar age could be due partly to food resource availability. A very small compensatory food intake was found in Octopus briareus when the density of food was kept constant (Borer, 1971). Physiological and behavioural compensation for dietary restrictions have been described in other molluscs (Survey and Rollo, 1991). Laboratory cultured cuttlefish, at a constant temperature and with plenty of good quality food, could be growing at the

1013

highest rate possible; compensatory mechanisms would not be possible. The IGR was highly correlated with different biochemical and morphometric parameters of S. oficinalis. The DGI showed no significant seasonal change in wild cuttlefish (Castro et al., 1991). In the present experience, a significant correlation was obtained between IGR and the DGI (Fig. l), showing that DGI may be used as an indicator of cuttlefish condition. Also, DGI increased rapidly (in 11 days), approaching the level of the control cuttlefish during the recovery period. These facts corroborate the short-term reserve function

2.5

2.0

.

RNA COR= 0.847

.

DNA COR50.055 .

-1

0

2

1

3

IGR Fig. 2. Variation of RNA, DNA and protein contents of cuttlefish mantle muscle with instantaneous growth rate (IGR). COR: correlation coefficient between variables. Points inside circles correspond to cuttlefish with significant growth after refeeding with thawed raw shrimp.

B. G. Castro and P. G. Lee

1014

Table 4. Total amino acid composition of proteins (NRC, 1982; Lentner, 1981) used for supplementation of surimi diets for cuttlefish and mean amino acid composition of ten cephalopods (Suyama and Kobayashi, 1980; Jhaveri et al., 1984; Iwasaki and Harada, 1985). n.a.: not analysed Amino acids Essential Leucine Isoleucine Valine Lysine Methionine Threonine Phenylalanine Tryptophan Arginine Histidine Non-essential Glycine Glutamate Aspartate Alanine Serine Cysteine Tyrosine Proline

Albumin (g/l6 g N) 8.2 5.6 7.8 :.;

Casein (g/l6 g N)

Whole egg (g/l6 g N)

Cephalopods (g/l6 g N)

4:8 5.8 1.8 5.4 2.2

10.2 5.6 7.4 8.2 2.9 5.0 5.4 1.4 4.0 3.0

8.3 5.6 7.5 6.2 3.2 5.1 5.1 1.8 6.1 2.4

8.0 5.2 4.6 7.7 3.2 4.7 3.9 n.a. 6.2 2.4

3.2 12.2 10.9 5.8 7.4 1.8 4.0 4.0

2.2 23.0 8.5 3.8 5.9 1.0 4.5 9.4

3.0 12.0 10.7 5.4 7.8 1.8 4.0 3.8

4.7 16.5 10.1 6.4 4.4 na. 3.8 5.8

of the digestive gland in cuttlefish (Castro et al., 1992). Therefore, DGI appears to be a good indicator of the recent nutritional state of cuttlefish. The CUT showed a strong negative correlation with IGR (Fig. 1). This fact would be expected because the cuttlebone is mainly mineral and it would not be resorbed during starvation, while the rest of the body would be depleted (Castro et al., 1992). Wild immature cuttlefish of approximately 500 g showed CUT values of 5.2% (Miramand and Bentley, 1992), corresponding to well fed cuttlefish growing with an IGR near 2% in this experiment. No data exist concerning the seasonal variation of CUT in wild cuttlefish, but it could be a better measure than the DGI for estimation of a wild cuttlefish’s condition during short periods. No changes in protein content (in fresh tissue) were found when IGR > 0, but there was an important decrease in protein with IGR < 0 (Fig. 2). This change was parallel to the variation in water content of the muscle. Mantle protein was shown not to decrease before 10 days of starvation (Castro et al., 1992). Thereafter, low content of protein in mantle muscle would indicate a poor condition in cuttlefish. Only

severe conditions, affecting the cuttlefish during a long period, would be reflected in a significant decrease in mantle protein content. A similar drop in muscle protein has been shown in senescent octopuses (Tait, 1986). However, all cuttlefish used in this experiment were immature or maturing such that mantle protein content may be useful as an indicator of long-term nutritional deficiencies in cuttlefish. The RNA/DNA ratio has been used as an indicator of growth in many species (Packard and Albergoni, 1970; Bullow, 1992). The 1987; Frantzis et al., RNA/DNA ratio increases significantly with growth rate at 17.5”C but not at 12.5”C in juvenile S. o#kinalis (Clarke et al., 1989). The RNA/protein ratio was shown to be correlated with the rate of protein deposition and fractional protein synthesis in mantle and other tissues of Octopus vulgaris (Houlihan et al., 1990). In the present experiment, both DNA and protein contents of mantle did not change significantly with growth (see Fig. 2), although the significant correlation of RNA/DNA ratio and RNA/protein ratios were due to the RNA change. Mantle RNA concentration increased to the level of the cuttlefish fed raw shrimp during the

Growth and condition in cuttlefish

ery period. Mantle RNA concentration should be useful as a short-term but not as a long-term indicator of growth and condition in cuttlefish. The DNA concentration per cell is considered constant within a species. The RNA concentration in tissues is associated with the potential capacity for protein synthesis in the tissue (Houlihan, 1991). Because RNA concentration in a tissue can be affected by the cell number, RNA content is divided by DNA content to estimate the RNA per cell (Bullow, 1987). However, mantle muscular tissue can have multinuclear cells altering this estimation of RNA by the cell, and the RNA activity can change, producing different rates of protein synthesis. Growth can occur either by an increase in the number of cells, increasing or not the amount of DNA, or in their sizes, with the DNA constant. Moreover, the RNA/DNA ratio estimates the synthetic processes and not the degradative ones associated with growth (Miglavs and Jobling, 1989). Therefore, the RNA/DNA ratio and, similarly, the RNA/protein ratio may not always be the best indicators of growth (Frantzis et al., 1992). Their use will depend on the tissue, individual size, species, life cycle stage and environmental factors (Bullow, 1987). In summary, protein and lipid supplemented surimi diets can be used as semipurified diets for growth and nutritional studies in cuttlefish, but growth rates will probably be low until more is understood of the cuttlefish’s amino acid requirements. Acknowledgements-This work was supported by a grant of the Ministerio de Education y Ciencia of the Spanish Government (to B. G. Castro), a United States National Institutes of Health DHHS grant (RR 04226-05) and the Marine Medicine general budget account of the Marine Biomedical Institute, University of Texas Medical Branch, Galveston, Texas. The authors thank F. P. DiMarco and R. H. DeRusha for their technical assistance.

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Borer K. T. (1971) Control of food intake in Octopus

1015

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