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Growth and energy utilization of juvenile pink abalone Haliotis corrugata fed diets containing different levels of protein and two starch:lipid ratios.

ABSTRACT Juvenile pink abalone Haliotis corrugata (initial mean length = 10.7 [+ or -] 0.3 mm; initial mean weight = 0.15 g) were grown under laboratory conditions in an aerated flow-through seawater system at 21 [+ or -] 1[degrees]C. For 131 days, abalone were fed diets containing three different levels of protein (42%, 36% and 32%), each level containing two ranges of starch:lipid ratios (1.5-1.8 or 3.2-3.6). The gross energy content of the diets ranged from 4.26-3.68 kcal/g. Within a particular protein level, growth did not differ significantly. When dietary protein levels exceeded 32%, a trend of higher growth of abalone was observed for dietary treatments that contained lower levels of lipid (higher starch to lipid ratios). The amount of protein consumed daily for the 32% dietary protein is apparently insufficient to meet requirements for energy and maximum growth. No notable diet-dependent differences in the proportional composition (dry weight) of the shell and the soft tissue, and the energy content of the soft tissue were observed. Daily food intake and consumed energy were significantly different among treatments, being inversely related to the calculated P:E ratios. The increased consumption may have been somewhat overestimated because of loss arising from lower water stability of the diets. Ammonia excretion ranged from 7.9-4.8 [micro]g N[H.sub.4.sup.+]/h/g abalone but was not significantly different among treatments except for the treatment containing 32% crude protein and a low carbohydrate:lipid ratio. Specific Dynamic Action (SDA) comprised nearly 50% of measured oxygen consumption and did not significantly differ among treatments. Respiration increased during the night showing a typical circadian pattern reported in Haliotis genera. Measured mucus production did not vary among treatments. Carbohydrate preferentially serves as the principal energy source and a consistent amount of dietary protein is also used as an energy source, regardless of dietary protein content. Within a developed energy budget, approximately 70% of the ingested energy was lost through feces, and approximately 25% of the ingested energy was metabolized, with 7% to 10% channeled to growth. A direct determination of available digestible energy was not possible because of the inability to collect sufficient feces to estimate fecal energy. The results suggest that practical diets should include levels of dietary protein that are approximately 35% protein to meet requirements for energy and maximum growth. In addition, carbohydrates seem to be the preferred energy source and lipid levels should be minimized to the extent that essential fatty acids requirements are met.

KEY WORDS: abalone, starch:lipid ratio; energy utilization, Haliotis corrugata

INTRODUCTION

Abalone aquaculture is a growing commercial enterprise and nutritionally balanced, formulated feed is becoming increasingly important as a substitute for their natural diet of macroalgae. The need for an effective formulated feed is based on not only achieving better growth performances but also avoiding the unpredictable availability of macroalgae. Production of commercial size abalone can take several years, because their nutritional requirements and physiology are yet to be fully understood. Thus, research on abalone nutritional requirements is important to increase efficiency through a combination of higher growth rates and lower costs.

Most animals consume their food to fulfill their energy requirements, and abalone is not an exemption (Gomez-Montes et al. 2003); it has been reported that abalone grow better when fed a formulated diet containing a protein:energy (P:E) ratio of approximately 100.

H. fulgens requires between 59-67 cal/g abalone/day to achieve maximum growth, at a crude protein intake of 6.22 [+ or -] 0.17 mg/g abalone/day with a protein efficiency ratio (PER) of 3.41 (Gomez-Montes el al. 2003). At higher intakes of protein, PER decreases significantly. For the best dietary P:E ratio, energy derived from nonprotein sources was satisfactory to minimize the use of protein as an energy source. For abalone feeding on a diet with a P:E ratio of 108, more dietary protein was used as a source of energy.

To achieve maximum growth, the deposition of dietary protein in the muscle tissue in abalone must be maximized and a proper balance of appropriate sources of dietary protein and energy will help to achieve this objective. Research conducted with different species of fish has consistently demonstrated an insufficient level of dietary protein yields lower growth rates (Smith 1989), and if the dietary level of energy in the diet is insufficient, then protein is used as a source of energy for maintenance (NRC 1983). A diet with the most effective P:E ratio will allow for a reduction in the level of dietary protein without a corresponding reduction in growth.

A modification of the relative proportions of dietary carbohydrate and lipid sources of energy may contribute to an even greater growth response. The dietary lipid requirement of abalone is believed to be low (Mai et al. 1995, Durazo-Beltran et al. 2003); therefore, carbohydrates are probably the preferred source of energy (Knauer et al. 1996, Monje & Viana 1998). A possible reduction in dietary lipid in response to the efficient use of carbohydrates as energy sources must be approached carefully to ensure that satisfaction of the essential fatty acid requirements is not compromised. A wide range of dietary starch levels seem to have no adverse effect on the growth of abalone, and cornstarch is considered to be a good carbohydrate source to balance the energy content of experimental diets (Knauer et al. 1996). Therefore, levels of carbohydrates can be changed to produce different dietary P:E ratios.

The objective of this work was to understand how dietary energy is used when pink abalone Haliotis corrugata are fed diets containing different dietary starch:lipid ratios and different protein levels.

MATERIALS AND METHODS

Diet Preparation

Six experimental diets were formulated (Table 1) to contain 3 different protein levels (42%, 36% and 32%) and two different starch:lipid ratio levels, ranging from either 1.5-1.8 and from 3.2-3.6 whereas also trying to maintain the P:E ratios (mg protein: kcal) between 90 and 100. Primary sources of protein were menhaden fish meal (EWOS) and isolated soybean protein (92% CP), ingredients were maintained at a 2:1 ratio to ensure a common dietary amino acid profile that generally reflected the proportional composition of soft tissue for abalone (Fleming et al. 1996). Because the dietary protein level was reduced, cornstarch was added to maintain the desired P:E ratio of the diets. Kelp meal was used as a filler as needed. The structural carbohydrates that constitute a high proportion of this ingredient were not included in the calculations of the starch to lipid ratios, because it is unknown whether these specific carbohydrates can be efficiently digested.

Each dietary treatment contained cod liver oil, which was added proportionally (from 6.3% to 3.1%) for each treatment. The composition and amounts of vitamin and mineral premixes that were added followed the recommendations of Hahn (1989). All ingredients were mixed thoroughly to produce a homogeneous mixture; water was added to achieve a moisture content of approximately 60%. The dough-like diets were cold extruded through a pasta machine (Rosito Bisani, Los Angeles) into 0.5 x 0.7 mm pellets, and then stored at -80[degrees]C until used. Water stability of each of the diets was determined in triplicate before and during measurements of intake by immersing 10 pellets of each diet within seawater held in control buckets (no animals inside). After 12 h of immersion, the amount of remaining feed (dry matter stability) was determined.

Proximate Analysis of Diets and Tissue

Proximate composition of diets and tissue was determined according to standard procedures (AOAC 1990). For diets, moisture content of each diet was calculated from triplicate samples (4-5g) that were dried to constant weight at 60[degrees]C. Mean total nitrogen content was determined by the microKjeldahl method, and percent crude protein was then calculated as % N x 6.25. Mean total dietary crude lipid was determined gravimetrically according to the method of Bligh & Dyer (1959). Fiber was determined by the acid-detergent method described in AOAC (1990). Mean ash content was determined by heating samples to 550[degrees]C for 4 h. The gross energy contents of diets and organisms (tissue and mucus) were determined by direct combustion in an adiabatic calorimeter Parr 1281. Nitrogen free extract was calculated by difference (NFE = 100 - [% crude protein + % crude lipid + % ash + % crude fiber]). Samples of the soft tissue of the abalone from each experimental unit were collected at the end of the experiment, frozen individually at -80[degrees]C and subsequently submitted to the same analysis.

Experimental Procedure

Three weeks prior to the initiation of the experiment, juvenile pink abalone H. corrugata were fed a diet consisting of diatoms (Navicula incerta and Nizchia closterium). Thereafter, for 1 week, a standard, nutritionally balanced formulated diet, containing fish meal, soybean meal, corn meal and kelp meal as the main ingredients (P:E ratio of 88) was fed as recommended by Durazo-Beltran et al. (2004). A total of 432 abalone with an average initial shell length and total weight of 10.70 [+ or -] 0.32 mm and 0.164 [+ or -] 0.01 g, respectively, were selected for use in the growth trial. Each experimental unit was a growing chamber that consisted of an ABS (acrylonitrile-butadiene-styrene) black pipe tube holding a container fitted with a plastic mesh floor (1 x 1 mm), and located at 3 cm from the pipe bottom. A total of 23 abalone were placed into each chamber that was held within a 4-L black bucket that was part of a flow-through system (250 mL/min). Each chamber was supplied with an air stone to maintain satisfactory levels of oxygen. Water temperature was maintained at 20[degrees]C [+ or -] 1[degrees]C throughout the feeding trial by a heat pump connected to an insulated 700-L reservoir, from which water was distributed to the growing chambers. Each treatment was run in triplicate for a total of 18 experimental units; treatments were randomly assigned to chambers in the culture system. A photoperiod of 12L/12D was maintained throughout the experiment, and diets were fed in excess every night at 20:00 hrs. On the following day at 08:00 h, any unconsumed diet was removed. Total feed intake (F) of each of the triplicate groups of abalone for each treatment was calculated twice during the last 8 days of each month. Daily dry matter loss from pellets in control chambers without abalone (stability) was used to adjust the feed intake that was estimated as:

F = (G [S/100]) - R (1)

where G represents the amount of feed offered, S is the amount of feed recovered from the control buckets, and R is the feed remaining in the containers with the experimental abalone.

The mean daily rate of feed intake for each treatment was then calculated as a percentage of body weight. Every 4 weeks, length and weight of all abalone within each replicate of each treatment were recorded as determined with an electronic digital caliper ([+ or -] 0.01 [micro]m) and an electronic scale (0.01 g error), respectively. The experiment was terminated after 131 days.

The following nutritional indices were calculated using the feed intake data.

Feed conversion efficiency,

FCE = 100 (wet weight gain [g])/(feed intake [g]) (2)

Protein efficiency ratio,

PER = (wet weight [g] gain)/(protein intake [g]) (3)

Specific growth rate (SGR, %/day) was estimated according to Hopkins (1992)

SGR = (ln [[w.sub.t]] - ln [[w.sub.i]])/t * 100 (4)

where ln ([w.sub.t]) is the natural logarithm of final body weight, ln ([w.sub.i]) is the natural logarithm of the initial body weight and t is the time in days.

Oxygen Consumption

Oxygen consumption was recorded using two computer-controlled polarographic oxygen sensors (Strathkelvin Instruments, Ireland), each with a capacity of six channels. Prior to use, the sensors were calibrated with a 0% oxygen solution (2% sodium sulfite in 0.01M sodium borate as buffer) and 100% saturated oxygen water. The sensors were held in the solution/water (21 [+ or -] 1[degrees]C) until variations were no longer recorded.

A block design with time (3 days) was used. The juvenile pink abalones were first acclimatized in smaller growing chambers for 20 h. The respective experimental diets were fed during the night (20:00 h), and during the following morning, the chambers were cleaned of any organic material that remained. At approximately noon of the day of an evaluation, the water flow of one unit of each treatment was closed and the chamber sealed for an incubation period of 50 min. The chamber was then opened for 10 min to restore the oxygen levels. This procedure was repeated 4 times. Two chambers were used as controls to determine the amount of oxygen consumed in the absence of abalone. The mean oxygen consumed in the control chambers was subtracted from the oxygen consumed by the experimental abalone. After each measurement, the organisms were then deprived of feed for 3 days and following the previously described procedure, oxygen consumption was measured to calculate the amount of energy used for maintenance (basal metabolism).

After termination of the incubation period, all abalone were removed from the chambers, blot dried with a piece of cloth, measured with digital calipers (MAX-CAL, [+ or -] 0.03 mm) at their longest dimension, and weighed using an analytic scale (AND SV-200, [+ or -] 0.01 g). Oxygen consumption rate (metabolic rate, V[O.sub.2]) was estimated from the corrected slope of the oxygen evolution curve (chambers with abalone minus chambers without abalone), after converting the % [O.sub.2] saturation to [micro]mol of dissolved [O.sub.2] in seawater, from known values of oxygen solubility (Green & Carritt 1967). The following equation was used for calculating metabolic rate:

V[O.sub.2e] = (Cs * m * 60)/(100% * n) (5)

where: V[O.sub.2e] = metabolic rate of the experimental organism (mL [O.sub.2]/g org/h); m = slope of the 02 evolution curve (%[O.sub.2]/min); 60 = factor used to transform from minutes to hours; Cs = Total amount of [O.sub.2] in the incubation chamber at 100% saturation ([micro]L [O.sub.2]); n = Number of organisms in the incubation chamber.

To determine the rate of oxygen consumption over a 24 h period, 80 organisms were randomly selected and equally divided among 4 chambers (20 per chamber). Oxygen consumption was measured continuously during successive 50 min periods in a closed system (static) followed by 10 min with open flow to recover the oxygen levels, as previously described. Four incubation chambers containing no abalone were used as replicate controls to determine the amount of oxygen that was not consumed by the abalone.

Ammonia Excretion and Pedal Mucus Production

Ammonia excretion was determined by introducing the abalone into 800-mL beakers containing Millipore-filtered and air-saturated seawater. To eliminate any nitrogen contamination, all glassware and beakers used were soaked with ammonia free detergent (alconox), then 5% hydrochloric acid, rinsed with distilled water, and stored in an N-free environment. Beakers were closed with a cover to seal the water and reduce the risk of N contamination during the incubation time. Control beakers, containing seawater but no animals, were incubated at the same time under the same conditions (20[degrees]C). Rates of ammonia excretion were determined as the ammonia content in the beakers with abalone minus the content in the control beakers after incubation at 30 and 60 min. Water samples were removed from the containers and kept at -80[degrees]C until processed in an automatic analyzer (SKALAR) by the Berthelot reaction and read at 640 nm. Rates of excretion were expressed as [micro]g N[H.sub.4.sup.+]/h/g abalone and converted to energy equivalents, using the conversion factor of 0.349 J/[micro]mol N-N[H.sub.4] (Widdows & Salkeld 1993).

To calculate the pedal mucus, abalone were placed in clean, dry 800-mL beakers that had been previously weighed. After 24 h, abalone were carefully removed and the feces in the beakers were washed away. The beakers were then dried and weighed, and the difference in weight estimated as mucus production (dry weight).

Statistical Analysis

To determine whether mean growth, expressed as final body weight (log transformed), fed the different diets were significantly different, a 2-way analysis of variance, 6 diets x 2 periods of 30 day each, was conducted. Diet and interaction were estimated using orthogonal contrasts. The daily feed intake based on measurements during the designated 16 days was calculated and compared among treatments using a 2-way (period and treatment) analysis of variance and multiple comparisons between least square means were performed using Tukey test when treatment showed a P < 0.05. Daily feed intake was used to estimate the amount of crude protein or calories consumed per g abalone. The calculated data was then analyzed as previously stated. FCE and PER of abalone of each treatment were calculated using the daily growth increments and the corresponding feed intake for the 16-day period, dry matter and protein content, respectively. All statistical analyses were performed using SAS-GLM procedures (SAS 8.2, 2001).

RESULTS

The analytically determined proximate composition and caloric content of the diets generally corresponded to the intended compositions and levels based on the formulations (Table 1). Mean stability of the diets representing the different treatments ranged from 88.4% to 77.6% and decreased with increasing levels of the kelp meal ingredient. Diets containing the highest levels of kelp meal had stabilities that were significantly less (P < 0.001) than those of all the other diets (Table 2). Feed intake, expressed as a percentage of body weight, was significantly greater for treatments [T.sub.6] (2.15%) and [T.sub.3] (2.21%) compared with treatments [T.sub.5] and [T.sub.2], (1.96%) and lowest for treatments [T.sub.4] and [T.sub.1] (1.64% and 1.67%, respectively). These values also showed an inverse trend relative to diet stability. Feed intake, expressed as calories/g abalone ranged between 84-70 and was inversely related to the stability of the diets. Feed intake, expressed as crude protein/g abalone, was not significantly different among treatments (Table 2), ranging from 7.0-7.4 mg CP/g abalone.

After 131 days (Table 2), within each of the two ranges of starch protein ratios, weight gain decreased as the P:E ratio decreased. For those diets with the highest protein levels, but the differences were not significant. Abalone fed the diets containing the lowest levels of protein and corresponding lowest P:E ratios had the lowest growth rates, significantly lower for those diets containing the high starch:lipid ratio. A similar response was observed for SGRs. The greatest FCEs were observed for abalone from treatments [T.sub.4] and [T.sub.1] (63.8 and 60.6, respectively). FCEs decreased as the water stability of the diets decreased. The somewhat lower levels of dietary fiber in treatments [T.sub.4] and [T.sub.1] may have contributed to the higher FCEs.

Highest PERs were associated with the diets that contained the highest levels of protein and the highest water stability. The PER for abalone from treatment [T.sub.4] (1.66) was significantly higher than that of abalone from each of treatments [T.sub.3] and [T.sub.2] (1.24 and 1.29, respectively) but not significantly different from those of abalone from the remaining treatments (Table 2). Generally, the proportional composition of shell/tissue of abalone from the different dietary among treatments was very similar except for abalone in treatment [T.sub.2] with a relatively higher and significant tissue proportion (Table 3).

No differences in rates of oxygen consumption of abalone were observed among dietary treatments with values ranging from 94.3-77.02 [micro]L [O.sub.2]/h/g abalone. In addition, the specific dynamic action (SDA) represented as the oxygen used for abalone to perform digestion for all dietary treatments was not significantly different and represented 52% to 67% (25.7-45.01 [micro]L [O.sub.2]/h/g abalone; calculated from Table 4) of the rate of total oxygen consumed.

Ammonia production of abalone ranged from 4.79-7.87 [micro]g N[H.sub.4]/h/g and were generally similar except for that values for abalone in dietary treatments [T.sub.1] and [T.sub.4] (7.61 and 7.87 N[H.sub.4]/h/g abalone, respectively) were significantly greater than that for abalone from treatment [T.sub.3] (4.79 N[H.sub.4]/h/g abalone). Metabolic rate remained relatively constant, with an average value of 90 [micro]L [O.sub.2]/h/g abalone W wt among treatments. The O:N ratio (molar) was similar, ranging from 9.0-10.2, for all dietary treatments with the exception of the significantly higher ratio for treatment [T.sub.3] (14.3). The higher ratio resulted from a comparatively lower rate of ammonia excretion. Mucus production, expressed as mg mucus/ day/g abalone and cal/day/g abalone, was not significantly different among treatments (statistics are not shown, however the percentages are given in as energy balance in Table 4).

As shown in the distribution of energy, expressed as a percentage of dietary intake (Table 4), energy loss caused by respiration ranged from 5.0% to 7.7% of total intake and SDA was quite similar among all dietary treatments, ranging from 6.7% to 8.5% of the total intake. Energy loss to ammonia production was comparatively low, between 0.52% and 1.03% for all treatments. Digestible energy values calculated as the sum of growth, respiration, ammonia and mucus production corresponded to one fourth of the feed intake with values ranging from 7.8-12.7 (cal/day). However, unexplained energy (including feces) counted for 73.8% to 79.2% from the feed intake.

Significant (P < 0.001) time-dependent differences in metabolism during night and day hours were observed for the juvenile abalone, reflecting a circadian rhythm (Fig. 1). The highest oxygen consumption was observed at night with values up to 120 [micro]L [O.sub.2]/h/g abalone W wt (22:00-04:00 h). The transition period was characterized by rapid changes in metabolic rates, such that abalone exhibited either a night- or light-adapted rate of metabolism. Percent survival remained above 90% throughout the experiment.

[FIGURE 1 OMITTED]

DISCUSSION

Although formulations of all diets were designed to achieve a P:E ratio that was within a range of 90-100, two of the actual ratios derived from the results of analytic determinations of nutrient composition for the 32% crude protein diets fell slightly below (87.5 and 88.3) the range. A trend of reduced weight gain as P:E ratios decreased was observed, and affirmed the observations reported by Gomez-Montes et al. (2003). Growth rates of H. corrugata are less than those reported for H. fulgens (Gomez-Montes et al. 2003), and this difference is confirmed from observations on a commercial farm (personal communication, BC Abalone farm Erendira Baja California, Mexico).

Values for feed intake were greater than those reported previously for Haliotis fulgens under the same experimental conditions (Gomez-Montes et al. 2003). Feed intake may have been overestimated for those diets that had the lowest water stability and caloric intake because all organisms were probably similar (approximately 70 cal [g.sup.-] abalone) within the different dietary treatments as suggested by the results previously observed for H. fulgens by Gomez-Montes et al. (2003). Nonetheless, caloric intake was generally higher than that found for H. fulgens at the same P:E ratio (Gomez-Montes et al. 2003). In addition, the calculations for FCE and PER for the diets with reduced water stability are probably overestimates, increasingly magnified as the water stability of an experimental diet decreased. This effect would most probably be enhanced by the disturbance and nutrient loss caused by the feeding activities of the live animals as the pellets become softer.

The amount of energy consumed was probably overestimated for those diets with reduced water stability, thereby affecting the calculation of the relative proportions of energy channeled to different metabolic activities. Therefore, the proportional losses of energy attributed to feces and unexplained energy (74% to 80%), are probably overestimates. Gomez-Montes et al. (2003) estimated feces and unexplained energy to represent approximately 45% to 55% of the total energy intake.

When dietary protein levels were approximately 36%, a trend of higher growth of abalone was observed for dietary treatments that contained lower levels of lipid (higher starch to lipid ratios). Previously published reports suggest that carbohydrates are preferentially used as an energy source during starvation (Durazo et al. 2004), whereas high levels of lipid (lower carbohydrate to lipid ratios) may actually retard growth (Durazo-Beltran et al. 2003). Despite sufficient carbohydrate being available as an energy source, a relatively stable amount of dietary protein is used to produce energy. Therefore, the dietary level of protein must be sufficient to satisfy the requirement for energy and still support maximum growth. Otherwise, maximum growth cannot be achieved. Maximum growth observed by Gomez-Montes et al. (2003) for H. fulgens fed diets with P:E ratios of 100 and 108 was probably because of provision of sufficient dietary protein (40% to 44%) to satisfy the protein derived energy requirement and to support maximum growth. The actual level of dietary protein needed would also be based on satisfaction of essential amino acid requirements.

Respiration rate serves as an accurate measurement of the metabolic state of animals. The lack of differences in respiration rate, both in basal metabolism and specific dynamic action (SDA), suggests that the physiologic processing of the nutrients in the diets is not different. The changes in oxygen consumption observed during the night appear to be typical of the entire Haliotis genera. The magnitude of this physiologic increase was greater than that previously reported for H. fulgens (Chacon et al. 2003), suggesting a possible species-specific characteristic.

Within a projected energy budget, more than 70% of the ingested energy was lost to feces or unexplained energy, and 7.2% to 9.9% was channeled to growth. In a previous investigation (Gomez-Montes et al. 2003), the proportion of consumed energy that was channeled to growth was higher and ranged from 17.8% to 22.8% for comparable P:E ratios. Nonetheless, the percent of digestible (not metabolizable) energy, expressed as the sum of growth, ammonia production, mucus production, SDA and basal metabolism, that was channeled to growth for the dietary treatments in our study (33.0% to 40.6%) was comparable to the values (32.6% and 44.6%) determined in the study of Gomez-Montes et al. (2003) with a diet of a similar P:E ratio fed to H. fulgens. However, in contrast to the latter study, digestible energy, estimated as a sum of growth, respiration, ammonia production and mucous production, composed a much lower proportion of the intake energy.

The inability to determine fecal energy in our investigation prevented a direct estimate of available digestible energy. In the determination of the proportional loss and use of ingested energy, digestibility is an important evaluation to estimate the amount of digestible energy. Previously the diet digestibility of H. fulgens was estimated (Gomez-Montes et al. 2003). However, collection of sufficient amounts of feces for proper analysis and confidence in results is difficult, and such amounts were not obtained in our study. Therefore, digestible energy was calculated as the sum of all expenditures, including energy loss caused by ammonia and mucus production. The proportional amount of digestible energy loss is much lower than that calculated for H. fulgens and is probably the result of the presumed overestimation of feed intake and corresponding energy intake.

The collective results suggest that dietary carbohydrates and proteins are the primary and secondary sources for abalone to suitably satisfy energy requirements. The dietary protein level must be sufficient to adequately meet requirements for energy and growth and the results of this experiment suggest that a protein requirement of approximately 35% will be sufficient, provided requirements of all essential amino acids are appropriately satisfied.

The range of lipid levels included in our experimental diets for both starch:lipid ratios probably exceeded the level needed to achieve maximum growth when sufficient protein is provided. When the protein level is met, then attention should be directed at reducing the total lipid level in the form of triglycerides to no more than 2% to 3%. The lipid requirement may be almost exclusively based on satisfaction of requirements of essential fatty acids. However, the possible growth enhancing effect of phospholipids needs to be investigated. Van Barneveld et al. (1998) reported that high levels of dietary lipids could negatively impact protein digestibility, but this relationship is not supported by data obtained in our investigation. The lack of a need for high levels of dietary triglycerides is noteworthy and should eliminate a potential problem of high levels of dietary lipids interfering with the successful manufacture of practical diets. An understanding of the digestibility of structural carbohydrates would also assist in the formulation of practical diets. Any future investigations of energy budgets of abalone must incorporate methods to ensure that all diets have strong and equivalent physical integrity so that more accurate determinations of the allocation of energy can be calculated.

ACKNOWLEDGMENTS

The authors thank BC Abalone farm for their kind donation of the abalone used in our experiment and Roche for donating the vitamin and mineral mixtures. This work was supported by the National Council for Science and Technology (CoNaCyT), Project G28119B.

LITERATURE CITED

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Bligh, E. G. & W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917.

Chacon, O., M. T. Viana, A. Farias, C. Vasquez & Z. Garcia-Esquivel. 2003. Circadian metabolic rate and short-term, response of juvenile green abalone (Haliotis fulgens Philippi) to three anesthetics. J. Shellfish Res. 22:415-422.

Durazo-Beltran, E., M. T. Viana, L. R. D'Abramo & J. F. Toro-Vazquez. 2003. Effect of triacylglycerols in formulated diets on growth and fatty acid composition in tissue of green abalone (Haliotis fulgens). Aquaculture 224(1-4):257-270.

Durazo, E., M. T. Viana, L. R. D'Abramo & J. Toro-Vazquez. 2004. Effects of starvation and dietary lipid on the lipid and fatty acid composition of muscle tissue of juvenile green abalone (Haliotis fulgens). Aquaculture 238:328-341.

Fleming, A. E., R. J. Van Barneveld & P. W. Hone. 1996. The development of artificial diets for abalone: A review and future directions. Aquaculture 140:5-63.

Gomez-Montes, L., Z. Garcia-Esquivel, L. R. D'Abramo, A. Shimada, C. Vasquez-Pelaez & M. T. Viana. 2003. Effect of dietary protein:energy ratio on intake, growth and metabolism of juvenile green abalone Haliotis fulgens. Aquaculture 220:769-780.

Green, E. & D. Carritt. 1967. New tables for oxygen saturation of seawater. J. Mar. Res. 25:140-147.

Hahn, K. O. 1989. Nutrition and growth of abalone. In: K. O. Hahn, editor. CRC Handbook of Culture of Abalone and Other Gastropods. Boca Raton, FL: CRC Press. pp. 135-156.

Knauer, J., P. J. Britz & T. Hecht. 1996. Comparative growth performance and digestive enzyme activity of juvenile South African abalone, Haliotis midae, fed on diatoms and practical diet. Aquaculture 140:75-85.

Mai, K., J. P. Mercer & J. P. Donlon. 1995. Comparative studies on the nutrition of two species of abalone, Haliotis tuberculata L. and Haliotis discus hannai Ino. III. Response of abalone to various levels of dietary lipid. Aquaculture 134:65-80.

Monje, H. & M. T. Viana. 1998. The effect of cellulose on the growth and cellulolytic activity of abalone Haliotisfulgens when used as an ingredient in formulated artificial diets. J. Shellfish Res. 17(3):667-672.

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Van Barneveld, R. J., A. E. Fleming, M. A. Vandeeper, J. A. Kruk & P. W. Hone. 1998. Influence of dietary oil type and oil inclusion level in manufactured feeds on the digestibility of nutrients by juvenile greenlip abalone (Haliotis laevigata). J. Shellfish Res. 17(3):649-655.

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JESSICA MONTANO-VARGAS, (1) MARIA TERESA VIANA, (2), * LOUIS R. D'ABRAMO, (3) ARMANDO SHIMADA (4) AND CARLOS VASQUEZ-PELAEZ (5)

(1) Programa de Maestria y Doctorado en Oceanografia Costera, Facultad de Ciencias Marinas, Universidad Autonoma de Baja California (UABC), Ensenada BC, Mexico; (2) Instituto de Investigaciones Oceanologicas, UABC, PO Box 453, Ensenada BC 22860, Mexico; (3) Department of Wildlife and Fisheries, Mississippi State University, PO Box 9690, Mississippi 39762; (4) Laboratorio de Rumiologia y Metabolismo Nutricional, Facultad de Estudios Superiores Cuautitlan, Universidad Nacional Autonoma de Mexico (UNAM), Juriquilla, Qro., Mexico; (5) Coordinacion de la Investigacion Cientifica. Facultad de Medicina Veterinaria y Zootecnia, UNAM, Ciudad Universitaria, DF, Mexico

* Corresponding author. E-mail: viana@uabc.mx
TABLE 1.
Ingredient (% dry weight), proximate composition
and energy content of six experimental diets
at two starch:lipid (S:L) ratios and three
protein levels.

Ingredients Dietary Treatments

 [T.sub.1] [T.sub.2] [T.sub.3]
Starch:Lipids (a) 1.5 21.8 31.6

Fish meal (b) 29.50 23.00 16.60
Soybean protein isolated (c) 14.75 11.50 8.30
Kelp meal (d) 17.29 29.94 42.54
Fish oil 6.30 5.70 5.10
Cornstarch 17.50 15.20 12.80
Common ingredients (e) 14.66 14.66 14.66

Proximate composition
Dry matter (%) 65.47 68.28 67.51
Crude protein (%) 42.06 35.93 32.13
Total lipids (%) 11.47 8.35 8.05
Crude fiber (%) 2.39 3.14 4.07
Ash (%) 15.66 19.59 23.21
NFEf 28.57 32.99 32.54
Energy (Kcal [g.sup.-1]) 4.26 4.00 3.67
P:E 98.70 90.00 87.50

Ingredients Dietary Treatments

 [T.sub.4] [T.sub.5] [T.sub.6]
Starch:Lipids (a) 43.3 53.2 63.6

Fish meal (b) 31.40 25.00 18.30
Soybean protein isolated (c) 15.70 12.50 9.15
Kelp meal (d) 11.24 24.24 37.79
Fish oil 3.70 3.40 3.10
Cornstarch 23.30 20.20 17.00
Common ingredients (e) 14.66 14.66 14.66

Proximate composition
Dry matter (%) 65.74 67.73 67.58
Crude protein (%) 42.64 37.57 32.48
Total lipids (%) 7.11 6.36 4.67
Crude fiber (%) 1.32 3.43 4.05
Ash (%) 13.54 17.72 21.92
NFEf 35.39 34.92 36.88
Energy (Kcal [g.sup.-1]) 4.26 3.88 3.68
P:E 100.10 96.80 88.30

TABLE 2.
Biological indices obtained for juvenile pink abalone
(Haliotis corrugata), feed diets at two starch:lipid
(S:L) ratios and three protein levels.

 Dietary Treatments

Starch: [T.sup.1]
Lipids (a) 1.5

Stability (%) 84.45 [+ or -] 0.61 (b)
Intake (%BW) 1.67 [+ or -] 0.03 (c)
Intake (cal
 [g.sup.-1] abalone) 71.54 [+ or -] 1.18 (cd)
Intake (mg PC
 [g.sup.-1] abalone) 7.03 [+ or -] 0.121
FCE (%) 60.57 [+ or -] 5.03 (a)
PER 1.42 [+ or -] 0.07 (a)
Weight change
 (mg [d.sup.-1]) 2.87 [+ or -] 0.23 (ab)
Weight gain (%) 226.52 [+ or -] 17.56 (ab)
SGR (% [d.sup.-1]) 0.90 [+ or -] 0.04 (ab)

 Dietary Treatments

Starch: [T.sup.2]
Lipids (a) 1.8

Stability (%) 82.55 [+ or -] 0.61 (b)
Intake (%BW) 1.96 [+ or -] 0.03 (b)
Intake (cal
 [g.sup.-1] abalone) 78.36 [+ or -] 1.18 (b)
Intake (mg PC
 [g.sup.-1] abalone) 7.04 [+ or -] 0.12 (a)
FCE (%) 45.61 [+ or -] 4.59 (ab)
PER 1.29 [+ or -] 0.07 (a)
Weight change
 (mg [d.sup.-1]) 3.09 [+ or -] 0.23 (ab)
Weight gain (%) 225.65 [+ or -] 17.5 (ab)
SGR (% [d.sup.-1]) 0.90 [+ or -] 0.04 (ab)

 Dietary Treatments

Starch: [T.sup.3]
Lipids (a) 1.6

Stability (%) 77.63 [+ or -] 0.62 (c)
Intake (%BW) 2.21 [+ or -] 0.03 (a)
Intake (cal
 [g.sup.-1] abalone) 84.05 [+ or -] 1.18 (a)
Intake (mg PC
 [g.sup.-1] abalone) 7.26 [+ or -] 0.12 (a)
FCE (%) 38.06 [+ or -] 4.59 (b)
PER 1.24 [+ or -] 0.07 (b)
Weight change
 (mg [d.sup.-1]) 2.29 [+ or -] 0.23 (b)
Weight gain (%) 196.05 [+ or -] 17.56 (b)
SGR (% [d.sup.-1]) 0.82 [+ or -] 0.04 (b)

 Dietary Treatments

Starch: [T.sup.4]
Lipids (a) 3.3

Stability (%) 88.43 [+ or -] 0.62 (a)
Intake (%BW) 1.64 [+ or -] 0.03 (c)
Intake (cal
 [g.sup.-1] abalone) 69.90 [+ or -] 1.18 (d)
Intake (mg PC
 [g.sup.-1] abalone) 7.00 [+ or -] 0.12 (a)
FCE (%) 63.83 [+ or -] 4.59 (a)
PER 1.66 [+ or -] 0.07 (a)
Weight change
 (mg [d.sup.-1]) 3.72 [+ or -] 0.23 (a)
Weight gain (%) 276.52 [+ or -] 17.56 (ab)
SGR (% [d.sup.-1]) 1.00 [+ or -] 0.04 (ab)

 Dietary Treatments

Starch: [T.sup.5]
Lipids (a) 3.2

Stability (%) 83.16 [+ or -] 0.61 (b)
Intake (%BW) 1.96 [+ or -] 0.03 (b)
Intake (cal
 [g.sup.-1] abalone) 76.25 [+ or -] 1.18 (bc)
Intake (mg PC
 [g.sup.-1] abalone) 7.38 [+ or -] 0.12 (a)
FCE (%) 53.03 [+ or -] 4.59 (ab)
PER 1.41 [+ or -] 0.07 (ab)
Weight change
 (mg [d.sup.-1]) 3.48 [+ or -] 0.23 (a)
Weight gain (%) 297.06 [+ or -] 17.56 (a)
SGR (% [d.sup.-1]) 1.04 [+ or -] 0.04 (a)

 Dietary Treatments

Starch: [T.sup.6]
Lipids (a) 3.6

Stability (%) 79.90 [+ or -] 0.65 (c)
Intake (%BW) 2.15 [+ or -] 0.03 (a)
Intake (cal
 [g.sup.-1] abalone) 80.09 [+ or -] 1.18 (ab)
Intake (mg PC
 [g.sup.-1] abalone) 7.08 [+ or -] 0.12 (a)
FCE (%) 39.32 [+ or -] 4.59 (b)
PER 1.35 [+ or -] 0.07 (ab)
Weight change
 (mg [d.sup.-1]) 2.28 [+ or -] 0.23 (b)
Weight gain (%) 194.85 [+ or -] 17.56 (b)
SGR (% [d.sup.-1]) 0.82 [+ or -] 0.04 (b)

(a) Starch:lipid corn starch added:lipid content
ratio. Standard errors are given.

Values in the same row with different superscripts
are statistically different P < 0.05.

TABLE 3.
Characteristics of shell and soft tissue for juvenile pink
abalone (Haliotis corrugata), fed diets at two starch:lipid (S:L)
ratios and three protein levels.

 Dietary Treatments

Dietary Treatments [T.sub.1]

Starch:Lipids (a) 1.5
Shell (% dry
 basis weight) 74.25 [+ or -] 1.47 (ab)
Soft tissue
 (% dry bais weight) 25.75 [+ or -] 1.47 (ab)
Dry soft tissue
 (% of live weight) 12.57 [+ or -] 0.53 (b)
Energy (Kcal [g.sub.-1]
 soft tissue) 4.83 [+ or -] 0.05 (a)

 Dietary Treatments

Dietary Treatments [T.sub.2]

Starch:Lipids (a) 1.8
Shell (% dry
 basis weight) 69.46 [+ or -] 0.85 (b)
Soft tissue
 (% dry bais weight) 30.53 [+ or -] 0.85 (a)
Dry soft tissue
 (% of live weight) 15.92 [+ or -] 0.30 (a)
Energy (Kcal [g.sub.-1]
 soft tissue) 4.82 [+ or -] 0.04 (a)

 Dietary Treatments

Dietary Treatments [T.sub.3]

Starch:Lipids (a) 1.6
Shell (% dry
 basis weight) 75.54 [+ or -] 0.85 (a)
Soft tissue
 (% dry bais weight) 24.46 [+ or -] 0.85 (b)
Dry soft tissue
 (% of live weight) 13.31 [+ or -] 0.30 (b)
Energy (Kcal [g.sub.-1]
 soft tissue) 4.75 [+ or -] 0.04 (a)

 Dietary Treatments

Dietary Treatments [T.sub.4]

Starch:Lipids (a) 3.3
Shell (% dry
 basis weight) 74.66 [+ or -] 0.85 (a)
Soft tissue
 (% dry bais weight) 25.34 [+ or -] 0.85 (b)
Dry soft tissue
 (% of live weight) 13.83 [+ or -] 0.30 (b)
Energy (Kcal [g.sub.-1]
 soft tissue) 4.78 [+ or -] 0.04 (a)

 Dietary Treatments

Dietary Treatments [T.sub.5]

Starch:Lipids (a) 3.2
Shell (% dry
 basis weight) 74.19 [+ or -] 0.85 (a)
Soft tissue
 (% dry bais weight) 25.80 [+ or -] 0.85 (b)
Dry soft tissue
 (% of live weight) 14.06 [+ or -] 0.30 (b)
Energy (Kcal [g.sub.-1]
 soft tissue) 4.85 [+ or -] 0.04 (a)

 Dietary Treatments

Dietary Treatments [T.sub.6]

Starch:Lipids (a) 3.6
Shell (% dry
 basis weight) 75.74 [+ or -] 0.85 (a)
Soft tissue
 (% dry bais weight) 25.24 [+ or -] 0.85 (b)
Dry soft tissue
 (% of live weight) 13.25 [+ or -] 0.30 (b)
Energy (Kcal [g.sub.-1]
 soft tissue) 4.74 [+ or -] 0.05 (a)

(a) Starch:lipids corn starch added:lipid content
ratio. Standard errors are given.

Values in the same row with different superscripts
are statistically different P < 0.05.

TABLE 4.
Energy budget for juvenile pink abalone (Haliotis corrugata) fed with
a balanced diet at two starch:lipid (S:L) ratios and three protein
levels. Energy balance is given per organism in relation to energy
intake.

 Dietary Treatments

 [T.sub.1]
 1.5
 Starch:Lipids (a) %

Intake (cal [day.sup.-1]) 38.90 100.00
Weight change (cal [d.sup.-1]) 3.34 8.58
SDA (cal [day.sup.-1]) (1) 3.09 7.94
Maintenance (cal [d.sup.-1]) (2) 2.81 7.22
Ammonia (cal [d.sup.-1]) 0.38 0.98
Mucus (cal [d.sup.-1] [g.sup.-1]) 0.48 1.23
Digestible energy (3) 10.1
Feces and unexplained energy 28.8 74.03
O:[N.sub.2] (molar) 9.4 [+ or -] 1.11 (b)
MR ([micro]L [O.sub.2] [h.sup.-1]
 [g.sup.-1] abalone) (4) 94.30 [+ or -] 6.97 (a)
MR ([micro]L [O.sub.2] [h.sup.-1]
 [g.sup.-1] abalone) (5) 49.29 [+ or -] 1.89 (a)
Ammonia ([micro]g N[H.sub.4.sup.+]
 [h.sup.-1] [g.sup.-1] abalone) 7.61 [+ or -] 0.44 (a)

 Dietary Treatments

 [T.sub.2]
 1.8
 Starch:Lipids (a) %

Intake (cal [day.sup.-1]) 45.79 100.00
Weight change (cal [d.sup.-1]) 4.55 9.94
SDA (cal [day.sup.-1]) (1) 3.07 6.70
Maintenance (cal [d.sup.-1]) (2) 2.83 6.18
Ammonia (cal [d.sup.-1]) 0.34 0.74
Mucus (cal [d.sup.-1] [g.sup.-1]) 0.39 0.85
Digestible energy (3) 11.18
Feces and unexplained energy 34.61 75.58
O:[N.sub.2] (molar) 10.2 [+ or -] 1.21 (b)
MR ([micro]L [O.sub.2] [h.sup.-1]
 [g.sup.-1] abalone) (4) 87.88 [+ or -] 6.97 (a)
MR ([micro]L [O.sub.2] [h.sup.-1]
 [g.sup.-1] abalone) (5) 55.34 [+ or -] 1.89 (a)
Ammonia ([micro]g N[H.sub.4.sup.+]
 [h.sup.-1] [g.sup.-1] abalone) 6.42 [+ or -] 0.44 (ab)

 Dietary Treatments

 [T.sub.3]
 1.6
 Starch:Lipids (a) %

Intake (cal [day.sup.-1]) 38.22 100.00
Weight change (cal [d.sup.-1]) 2.66 6.96
SDA (cal [day.sup.-1]) (1) 2.87 7.51
Maintenance (cal [d.sup.-1]) (2) 1.89 4.95
Ammonia (cal [d.sup.-1]) 0.20 0.52
Mucus (cal [d.sup.-1] [g.sup.-1]) 0.32 0.84
Digestible energy (3) 7.95
Feces and unexplained energy 30.27 79.20
O:[N.sub.2] (molar) 14.3 [+ or -] 0.74 (a)
MR ([micro]L [O.sub.2] [h.sup.-1]
 [g.sup.-1] abalone) (4) 90.98 [+ or -] 6.97 (a)
MR ([micro]L [O.sub.2] [h.sup.-1]
 [g.sup.-1] abalone) (5) 54.94 [+ or -] 1.89 (a)
Ammonia ([micro]g N[H.sub.4.sup.+]
 [h.sup.-1] [g.sup.-1] abalone) 4.79 [+ or -] 0.44 (b)

 Dietary Treatments

 [T.sub.4]
 3.3
 Starch:Lipids (a) %

Intake (cal [day.sup.-1]) 46.69 100.00
Weight change (cal [d.sup.-1]) 4.51 9.70
SDA (cal [day.sup.-1]) (1) 3.94 8.47
Maintenance (cal [d.sup.-1]) (2) 3.19 6.86
Ammonia (cal [d.sup.-1]) 0.48 1.03
Mucus (cal [d.sup.-1] [g.sup.-1]) 0.54 1.16
Digestible energy (3) 12.66
Feces and unexplained energy 33.83 72.77
O:[N.sub.2] (molar) 9.0 [+ or -] 0.72 (b)
MR ([micro]L [O.sub.2] [h.sup.-1]
 [g.sup.-1] abalone) (4) 93.18 [+ or -] 6.97 (a)
MR ([micro]L [O.sub.2] [h.sup.-1]
 [g.sup.-1] abalone) (5) 51.52 [+ or -] 1.89 (a)
Ammonia ([micro]g N[H.sub.4.sup.+]
 [h.sup.-1] [g.sup.-1] abalone) 7.87 [+ or -] 0.44 (a)

 Dietary Treatments

 [T.sub.5]
 3.2
 Starch:Lipids (a) %

Intake (cal [day.sup.-1]) 46.61 100.00
Weight change (cal [d.sup.-1]) 4.35 9.33
SDA (cal [day.sup.-1]) (1) 3.56 7.64
Maintenance (cal [d.sup.-1]) (2) 3.58 7.68
Ammonia (cal [d.sup.-1]) 0.38 0.82
Mucus (cal [d.sup.-1] [g.sup.-1]) 0.34 0.73
Digestible energy (3) 12.21
Feces and unexplained energy 34.40 73.80
O:[N.sub.2] (molar) 10.1 [+ or -] 1.4 (b)
MR ([micro]L [O.sub.2] [h.sup.-1]
 [g.sup.-1] abalone) (4) 90.48 [+ or -] 6.97 (a)
MR ([micro]L [O.sub.2] [h.sup.-1]
 [g.sup.-1] abalone) (5) 50.60 [+ or -] 1.89 (a)
Ammonia ([micro]g N[H.sub.4.sup.+]
 [h.sup.-1] [g.sup.-1] abalone) 6.75 [+ or -] 0.44 (ab)

 Dietary Treatments

 [T.sub.6]
 3.6
 Starch:Lipids (a) %

Intake (cal [day.sup.-1]) 36.23 100.00
Weight change (cal [d.sup.-1]) 2.62 7.23
SDA (cal [day.sup.-1]) (1) 2.67 7.37
Maintenance (cal [d.sup.-1]) (2) 1.87 5.16
Ammonia (cal [d.sup.-1]) 0.26 0.72
Mucus (cal [d.sup.-1] [g.sup.-1]) 0.38 1.05
Digestible energy (3) 7.80
Feces and unexplained energy 28.43 78.47
O:[N.sub.2] (molar) 9.11 [+ or -] 0.41 (b)
MR ([micro]L [O.sub.2] [h.sup.-1]
 [g.sup.-1] abalone) (4) 77.02 [+ or -] 6.97 (a)
MR ([micro]L [O.sub.2] [h.sup.-1]
 [g.sup.-1] abalone) (5) 51.35 [+ or -] 1.89 (a)
Ammonia ([micro]g N[H.sub.4.sup.+]
 [h.sup.-1] [g.sup.-1] abalone) 6.35 [+ or -] 0.44 (ab)

(a) Starch:lipids; corn starch added:lipid content ratio.

(1) Calculated by difference between fed and unfed abalone.

(2) Respiration from abalone left under inanition for three days.

(3) Calculated as the sum of growth, respiration, ammonia and
mucus production.

(4) Oxygen consumption from fed abalone.

(5) Oxygen consumption from unfed abalone.

(a,b) Values in the same row with different
superscripts are statistically different P < 0.05.
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Author:Vasquez-Pelaez, Carlos
Publication:Journal of Shellfish Research
Date:Dec 1, 2005
Words:7845
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