Influence of conditioning diet and spawning frequency on variation in egg diameter for Greenlip abalone, Haliotis laevigata.

ABSTRACT Both nutrient provisioning of eggs, during development within the ovary, and egg diameter are believed to be important factors governing the development and survival of invertebrate larvae. The diet of female broodstock and the conditioning regimen are likely to influence these parameters. In this study we examine the effect nf broodstock diet (three formulated diets differing in levels of the fatty acid, Arachidonic acid [ARA], and a red seaweed diet) and spawning frequency on egg diameter variability both within a batch of eggs spawned from one female and between batches of eggs. Greenlip abalone broodstock were spawned at the beginning of the experiment and again every 16 wks (1131[degrees]C days effective accumulative temperature [EAT]) using commercial hatchery practices. The variability of egg diameter within batches spawned from the same female over three consecutive spawning rounds and within diet treatments was determined. Cytoplasm diameter, vitelline layer and jelly coat thickness of unfertilized eggs were also compared between eggs spawned from individual females and between females. In addition, the relationships between broodstock parameters (weight and shell length) and egg diameter were explored. Eggs spawned from females feeding on a red seaweed diet were smaller than eggs from the low ARA treatment. Depending on spawning frequency, broodstock diet can influence the cytoplasm diameter and jelly coat thickness. No relationship between egg diameter and broodstock parameters was found. However, batches spawned from the same female abalone became more variable over time with a shift in size frequency distribution. Results indicate that the variability of egg diameter within a batch changes from female to female and highlights the importance of selecting successful broodstock for conditioning at commercial hatcheries.

KEY WORDS: abalone, Haliotis laevigata, diets, egg diameter, arachidonic acid

INTRODUCTION

One of the crucial factors controlling the development of the Australian abalone aquaculture industry is the successful conditioning of broodstock because the supply of suitable wild broodstock has at times been limiting. Gonad conditioning provides flexibility in production cycles and more economical use of hatchery resources (Lleonart 1992) as well as allowing genetic improvement programs.

Abalone larvae are lecithotrophic and rely heavily on yolk reserves provided by the egg to fuel development (Jaeckle & Manahan 1989). Previous studies have indicated that smaller eggs have improved fertilization rates and contain more total lipid than larger eggs (Daume & Ryan 2004). However, it is widely considered for invertebrates, that larger eggs produce larger offspring with a greater chance of survival (Levitan 2000), and that fertilization is increased at lower sperm concentrations for larger eggs (Levitan 1996, Marshall et al. 2002).

It has been proposed that the most appropriate method of determining reproductive cycles in molluscs and echinoderms is to measure oocytes (Grant & Tyler 1983). However, egg diameter, post spawning, is believed to be a key determinant of reproductive performance (Brooks et al. 1997) and small differences in egg diameter are considered to have significant biological consequences (George 1999). The ratio of cytoplasm to egg diameter is considered to be useful for assessing reproductive performance in blacklip abalone, Haliotis rubra Leach, (Littay & De Silva 2001) and to provide a measure of optimal egg size for fertilization experiments without the need for histological examination.

Earlier research on broodstock conditioning with greenlip abalone (Haliotis laevigata, Donovan) showed evidence of nutrient depletion of eggs during lengthy conditioning periods and high spawning induction frequency (Daume & Ryan 2004, Freeman et al. submitted).

Thus nutrient provisioning of eggs and egg diameter are important factors governing the development and survival of larvae. The diet of female broodstock and the conditioning regimen are likely to contribute to the quality of abalone eggs. A crucial step in successful abalone aquaculture is selecting female broodstock that exhibit high fecundity and produce high quality eggs over consecutive spawnings. This reduces the need to hold large groups of females and minimizes the amount of hatchery resources, such as feed, needed to maintain the broodstock.

This study examined the effect of diet (three formulated diets with differing levels of the fatty acid, ARA and a red seaweed diet) and spawning frequency on the variability in egg diameter within a batch of eggs spawned from one female and between batches and is part of a larger study of the influence of these diets on fecundity, egg quality and larval success.

MATERIALS AND METHODS

Broodstock Conditioning

Greenlip abalone, Haliotis laevigata, were collected in August 2003 from Augusta (115[degrees]16'E; 34[degrees]32'S) and Hopetoun (120[degrees]13'E; 33[degrees]95'S), Western Australia and held indoors at Great Southern Marine Hatcheries, Albany, Western Australia.

Broodstock were randomly assigned to spawning groups (A to D), and within each spawning group there were 12 round 60 L plastic tubs of females and two tubs of males, with each tub containing 3 animals.

Broodstock were exposed to a photoperiod of 12L:12D at ca. 100 lux (measured at the bottom of tubs). The conditioning system received flow through, temperature controlled seawater (average temperature, 17 [+ or -] 0.2[degrees]C).

Broodstock (average length 132.5 [+ or -] 3.5 mm; average weight 352.2 [+ or -] 27.6 g) were spawned prior to treatment conditioning and again every 16 wks, (i.e., every 1131[degrees]C-days EAT). The study was conducted from October 2003 to October 2004 including a natural spawning season.

EAT was calculated using the formula (Kikuchi & Uki 1974):

[Y.sub.n] = [n.summation ove i=1] ([t.sub.i] - [theta])

where [Y.sub.n] ([degrees]C days) = EAT, n (days) = number of days since water temperature rose above [theta], [t.sub.i] ([degrees]C) = daily water temperature in which the animal was reared, and [theta] ([degrees]C) = biological zero point for gonad maturation (6.9[degrees]C for H. laevigata; Grubert & Ritar 2003). Spawning dates were October 2003 for the spawn out, January/February 2004 for the first, May/June 2004 for the second and September 2004 for the third spawning (Table 1).

Within each spawning group (Table 1), 36 females (9 females per treatment) and 6 male greenlip abalone were individually induced to spawn using a combination of desiccation for 1 h, heat stress at 21[degrees]C and UV treated seawater.

Diet

Female broodstock were conditioned on four different diets, with three replicated tubs (9 animals in total) per diet treatment and per spawning group. One set of females were fed a formulated diet with a low enrichment level of arachidonic acid (EPA: ARA = 3:8; 1% of total fatty acids), the second set was fed a formulated diet containing a high level of ARA (EPA: ARA = 1:16; 2% of total fatty acids). A third set was fed a formulated diet containing no arachidonic acid (EPA: ARA = 6:0, 0% of total fatty acids). The formulation of the diet is proprietary. The fourth set of females was fed a mixed red seaweed diet (e.g., Plocamium mertensii, Gracilaria sp.). The mixed red seaweed was collected from the Southern Ocean mainly around Albany (117[degrees]95'E; 34[degrees]90'S); the Gracilaria sp. was collected from Hopetoun (120[degrees]13'E; 33[degrees]95'S). Animals were cleaned and fed ad libitum daily. Feed intake was monitored daily.

Egg Measurements

Unfertilized egg samples were collected within 30 min of the female spawning and immediately fixed in 10% formalin. Thirty eggs were then measured using an eyepiece graticule on an inverted microscope. Cytoplasm diameter, vitelline layer and jelly coat thickness were compared between individual females, diet treatments and across consecutive spawnings (Fig. 1). The variability of egg diameter within batches spawned from the same female over three consecutive spawning rounds and within diet treatments were determined. Egg diameter includes the vitelline and cytoplasm components of the egg. The ratio of cytoplasm to egg diameter was calculated as the cytoplasm measurement divided by the egg diameter. Eggs were classed as primary eggs when the jelly coat and vitelline layer was absent and the total egg diameter was on average 111.51 [+ or -] 10.54 [micro]m.

Data Analysis

All data analyses were carried out using Statistica software (version 6.0. StatSoft, Inc. 2002). Normality of all data was checked graphically using boxplots and with the Kolmogorov-Smirnov test.

Comparisons of egg parameters (cytoplasm, vitelline layer and jelly coat thickness), and comparisons of broodstock parameters with batch and egg size, were carried out using analysis of variance (ANOVA) with Tukey post hoc comparisons. Repeated measure analyses could not be performed, because different individuals spawned successfully during each of the three attempted spawnings.

Weight and shell length data in relation to weight loss were investigated using repeated measures ANOVA and Tukey post hoc comparisons. The relationship between broodstock parameters (length and weight) and egg diameter were explored with a simple regression analysis (P < 0.05).

Size-frequency distribution of egg diameter were compared over time and between treatments where appropriate using descriptive statistics (minimum, maximum, size range, first and third quartile). Interquartile differences between the first and third quartile were used as a measure of spread of the size-frequency distributions.

RESULTS

Influence of Diets on Cytoplasm Diameter, Vitelline Layer and Jelly Coat Thickness, Egg Diameter and the Ratio of Cytoplasm to Egg Diameter of Abalone Eggs over Three Consecutive Spawnings

During the first spawning, diet had a significant effect on cytoplasm diameter and jelly coat thickness (df = 9, F = 2.13, P = 0.03), however there was no significant difference in vitelline thickness between treatments (Table 2). Eggs obtained from females fed the red seaweed diet were smaller in cytoplasm diameter than the low ARA treatment (post hoc = 0.005). Jelly coat diameter was largest in the red seaweed treatment and significantly larger than in the low ARA treatment (post hoc = 0.007).

Diet treatments were found to have no significant effect on cytoplasm diameter, vitelline layer or jelly coat thickness of eggs obtained from the second (df = 9, F = 1.102, P = 0.38) and third consecutive spawning (df = 9, F = 1.12, P = 0.35).

Across all spawnings, diet significantly influenced egg diameter (df = 3, F = 3.24, P = 0.023). Egg diameter in the red seaweed diet was significantly smaller than egg diameter in the low ARA (post hoc = 0.015), but did not differ significantly from the high ARA or control diet.

In all four diet treatments, the ratio of cytoplasm to egg diameter decreased between the first and third spawning rounds. The ratio was highest in the low ARA treatment during the first spawning round and decreased to 0.88 in the third spawning round. Similarly the ratio in the high ARA treatment decreased to 0.88 in the third round, from 0.92 in the first round and 0.90 in the second round.

The occurrence of primary eggs in batches spawned by females fed the red seaweed diet increased over the three consecutive spawning rounds (Table 2). However, primary eggs occurred more frequently in the three formulated diet treatments, particularly during the first spawning round.

Relationship Between Diet, Shell Length, Weight and Weight Change of Female Broodstock over Three Consecutive Spawning Rounds

Table 3 shows the shell length, weights and weight changes of all female broodstock at each individual spawning including the initial spawn out. Over the whole experimental period, all animals lost weight and time had a significant effect on weight loss (df = 3, F = 11.78, P = 0.00). Broodstock weight at spawnout was significantly higher than weight during the first (post hoc P < 0.001), second (post hoc = 0.000133) and third (post hoc P < 0.001) spawnings.

Size Frequency Distribution of Eggs Spawned by the Same Female over three Consecutive Spawning Rounds

Red Seaweed

Eggs spawned by female one during the first spawning ranged between 208-229 [micro]m, with 75% (3rd quartile) of the eggs being within 210 and 224 [micro]m in diameter (Table 4, Fig. 2a). The spread of data became slightly more variable during the second spawning increasing to an interquartile difference of 7.23 from 5.26 during the first and third spawning. For the second female, variability in egg size decreased from the first to the second spawning and then increased slightly during the third spawning (Interquartile difference 7.23, 2.63 and 5.92 respectively). The spread of data for the third female was more consistent over all three spawnings with the interquartile difference remaining at 5.26. Egg diameter ranged between 210-230 [micro]m.

High ARA

Eggs obtained from this female fed the high ARA formulated feed were less variable over time, with eggs ranging between 203-221 [micro]m during the first spawning and between 210-230 [micro]m and 210 and 229 [micro]m in the second and third spawnings, respectively (Table 4, Fig. 2b). The interquartile difference stayed constant at 5.26 during all three spawnings.

Low ARA

The size frequency distribution of eggs obtained from the first female were highly variable between the first and second spawnings, with the distribution shifting from between 200-253 [micro]m, respectively (Table 4, Fig. 2c). The interquartile difference increased from 2.63-15.78 from the first to the second spawning and decreased to 6.57 during the third spawning. The second female only spawned during the first and second spawning, however the size variability was smaller and ranged from 210-239 [micro]m.

Control Diet (No ARA)

During the first spawning round eggs obtained from the first female ranged between 204-228 wm with 75% (3rd quartile) of the eggs within 205 and 218 [micro]m in diameter (Table 4, Fig. 2d). The interquartile difference decreased slightly from 6.25-5.26 during the second spawning round, and was highest at 11.84 during the third spawning round. The eggs spawned by the second female were less variable during the first and third spawning. The interquartile difference was lowest during the first spawning (5.59) and increased to 7.89 and 7.56 during the second and third spawning respectively. The third female spawned during only the first and second spawning however, egg size decreased over these time periods with eggs ranging from 210-224 over the first and second rounds, respectively.

DISCUSSION

Influence of Diets on Broodstock Parameters and Egg Parameters

The results of this study suggest that diet is not the only factor controlling the size of the eggs produced. Over all three consecutive spawnings, egg diameter in the red seaweed treatment differed significantly from only the low ARA diet, and relative differences varied with spawning rounds. During the first spawning round, eggs spawned by females fed the red seaweed diet were significantly smaller in cytoplasm diameter and had significantly larger jelly coats than those in the low ARA treatment. However, during the second and third consecutive spawnings there was no significant effect of diet on cytoplasm diameter, vitelline layer thickness or jelly coat thickness. Previous studies have indicated that diet directly influences egg quality in marine invertebrates (Jaeckle 1995). On the other hand, Nevejan et al. (2003) found that the total lipid content of eggs and the size of the eggs spawned by the scallop, Argopecten purpuratus were independent of diet. Similarly Caers et al. (2002) found there to be no significant effect of diet on the egg size of the oyster, Crassostrea gigas.

The role of diet in this study is unclear. Whether the increase in egg diameter in the low ARA treatment is correlated with the ARA content of the eggs will be determined after subsequent biochemical analysis of the eggs. It is likely however that the maternal condition of the female broodstock during the second spawning was negatively influenced by some other factor like overall animal nutritional condition. All animals lost weight during the experiment particularly when feeding on the formulated diet at the start of the experiment. It could be suggested that animals needed a weaning period to get used to the new diets because weight loss was less apparent on the natural diet and decreased on the formulated diets after the first spawning.

If measurements of egg diameter are an accurate representation of egg ripeness, because larger eggs are more ripe than smaller eggs, then those eggs obtained from the low ARA formulated feed treatments were more ripe than those obtained from the red seaweed treatment. On the other hand the low ARA diet showed the highest percentage occurrence of primary eggs. It is possible that these larger eggs contained more moisture as found by Daume and Ryan (2004) and were thus not a true reflection of ripeness. It may also be that the micronutrient content of the formulated feeds may be lower than optimal. The red seaweed diet is a mixed species diet and subsequently may be lacking in some macronutrients. However, eggs in the red seaweed treatment showed the lowest percentage occurrence of primary eggs. A follow up study investigating combined broodstock conditioning diets, where broodstock are first fed a low ARA formulated diet and then finished off with red seaweed before spawning, is planned.

The decrease in the ratio of cytoplasm to egg diameter across all treatments may suggest that egg quality deteriorated over the consecutive spawnings. Littay and De Silva (2001) reported for blacklip abalone, that a ratio of yolk (equivalent to cytoplasm in this study) to total egg diameter between 0.83-0.87 was ideal for this species and provided the highest fertilization rate. These authors suggested that any deviation from this range (0.83-0.87) indicates overripe eggs or declining egg biochemical status. The ratios found in our study were much higher than those found by Littay and De Silva (2001) however that might be attributable to the different species investigated in this study.

Size Frequency Distribution of Eggs

The descriptive statistics of the size frequency distributions of eggs spawned from individual females suggest that the eggs within the gonad are not uniform in size and display high variability between consecutive spawnings. This may be directly related to conditioning time, with the broodstock needing a longer conditioning period to produce eggs of similar size and quality.

Huchette et al. (2004) found that development of blacklip abalone oocytes within the gonad was not uniform and resulted in eggs that were variable in size. Clavier (1992) suggested that only the largest and ripest oocytes within the gonad are released during a spawning. However, in this study egg size was very variable over single spawnings for some females. Broodstock may have responded to nutritional stress, resulting in resorption of ripe gametes. Martinez et al. (1992) concluded that a reduction in the gonad index of a hatchery-reared scallop, Argopecten purpuratus, was caused by resorption of ripe gametes because the broodstock were under nutritional stress.

In this study, egg diameter was measured as total egg diameter including the vitelline layer and the cytoplasm. However, according to Grant and Tyler (1983), the most appropriate method of determining reproductive cycles in molluscs and echinoderms is to measure oocytes. On the other hand, egg diameter is believed to be a key determinant of reproductive performance in fish (Brooks et al. 1997) and small differences in egg diameter are considered to have significant biological consequences (George 1999). Measuring egg diameter of unfertilized eggs (post spawning) is a relatively quick and simple means of gaining reproductive information about broodstock without the need of culling broodstock to measure oocytes.

CONCLUSION

In this study we showed that the egg diameter varied between eggs spawned from females (14. laevigatu) feeding on a red seaweed compared with the low ARA diet. Depending on spawning frequency, broodstock diet can also influence the cytoplasm diameter and jelly coat thickness. Egg parameter measurements and ratios of cytoplasm to egg diameter may be useful parameters for assessing reproductive performance in greenlip abalone. Studies are needed to further investigate the influence of broodstock diets on egg variability and ultimately on larval survival. Combination diets, where broodstock are fed a formulated feed for the better part of the conditioning period, and then a suitable red seaweed species will be tested.

ACKNOWLEDGMENTS

The authors thank Great Southern Marine Hatcheries, Australia for hosting this part of the study and their staff and Kylie Freeman, Mathieu Castex and Misty Shipway for helping with spawnings and abalone egg measurements. Drs Greg Maguire and Anthony Hart provided useful editorial input. This study was funded as part of an FRDC project (2003/203). Dr Brett Glencross formulated the broodstock diets and he and Wayne Hawkins produced the diets.

LITERATURE CITED

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Caers, M., S. D. Utting, P. Coutteau, P. Millican & P. Sorgeloss. 2002. Impact of the supplementation of a docosahexaenoic acid-rich emulsion on the reproductive output of oyster broodstock, Crassostrea gigas. Mar. Biol. 140:1157-1166.

Clavier, J. 1992. Fecundity and optimal sperm density for fertilization in the ormer (Haliotis turberculata L.). In: S. A. Shepherd, M. Tegner & S. Guzman del Proo, editors. Abalone of the world. Biology, fisheries and culture. Blackwell, Oxford: Blackwell Scientific Publications Inc. pp. 86-92.

Daume, S. & S. Ryan. 2004. Fatty acid composition of eggs derived from conditioned and wild caught greenlip abalone broodstock (Haliotis laevigam). J. Shellfish Res. 23(4):967-974.

Freeman, K., S. Daume, M. Rowe, G. Maguire, S. Parsons & R. Lambert. A broodstock conditioning trial with greenlip abalone (Haliotis laevigam Donovan) in Western Australia. J. Shellfish Res. (submitted). (same issue)

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Grubert, M. A. & A. J. Ritar. 2003. The effect of temperature and conditioning intervals on the spawning success of wild-caught blacklip (Haliotis rubra) and greenlip (H. laevigata) abalone fed an artificial diet. In: A. E. Fleming, editor. Proceedings of the 10th Annual Abalone Aquaculture Workshop, November 19-21, 2003. Port Lincoln, Australia. Abalone Aquaculture Subprogram, Fisheries Research and Development Corporation, Canberra, Australia.

Huchette, S. M. H., J. P. Soulard, C. S. Koh & R. W. Day. 2004. Maternal variability in the blacklip abalone, Haliotis rubra leach (Mollusca: Gastropoda): effect of egg size on fertilisation success. Aquaculture 231:181-195.

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Kikuchi, S. & N. Uki. 1974. Technical study on artificial spawning of abalone, genus Haliotis. I. Relation between water temperature and advancing sexual maturity of Haliotis discus hannai Ino. Bull. Tohoku Reg. Fish. Res. Lab. 33:69-78.

Levitan, D. R. 1996. Effects of gamete traits on fertilization in the sea and the evolution of sexual dimorphism. Nature 382:153-155.

Levitan, D. R. 2000. Optimal egg size in marine invertebrates: theory and phylogenetic analysis of the critical relationship between egg size and development time in echinoids. Am. Nat. 156(2): 175-192.

Lleonart, M. 1992. A gonad conditioning study of the greenlip abalone (Haliotis laevigata). University of Tasmania, Hobart. 162 pp.

Littay, M. & S. S. De Silva. 2001. Reproductive indices based on physical characteristics of female blacklip abalone Haliotis rubra L. J. Shellfish Res. 20(2):673-677.

Marshall, D. J., C. A. Styan & M. J. Keough. 2002. Sperm environment affects offspring quality in broadcast spawning marine invertebrates. Ecol. Lett. 5:173-176.

Martinez, G., M. Torres, E. Uribe, M. A. Diaz & H. Perez. 1992. Biochemical composition of broodstock and early juvenile Chilean scallop, Argopecten purpuratus Lamarck, held in two different environments. Z Shellfish Res. 11:307 313.

Nevejan, N., V. Courtens, M. Hauva, G. Gajardo & P. Sorgeloos. 2003. Effect of lipid emulsions on production and fatty acid composition of eggs of the scallop Argopecten purpuratus. Mar. Biol. 143:327-338.

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FIONA GRAHAM, (1) * TAHRYN MACKRILL, (1,2) MARK DAVIDSON (1,2) AND SABINE DAUME (1)

(1) Research Division, Department of Fisheries, Western Australia, PO Box 20, North Beach, WA 6920, Australia; (2) c/o Great Southern Marine Hatcheries, PO Box L34, Little Grove, Albany WA 6330, Australia

* Corresponding author. E-mail: [e-mail]

TABLE 1.

Spawning dates for each spawning group.

                              Spawning groups
    Spawning
     events           A          B          C          D

Spawn out          07.10.03   15.10.03   22.10.03   29.10.03
First spawning     26.01.04   03.02.04   11.02.04   18.02.04
Second spawning    19.05.04   23.05.04   31.05.04   08.06.04
Third spawning     08.09.04   14.09.04   22.09.04   29.09.04

TABLE 2.

Influence of diet on cytoplasm diameter, vitelline layer and jelly coat
thickness of abalone, Haliotis laevigata, eggs and the presence of
primary eggs over three consecutive spawning periods (1, 2 and 3).
(Data are means ([micro]m) [+ or -] SE)

           Spaw-
   Diet    ning              Cytoplasm                 Vitelline

Red          1     194.39 [+ or -] [1.52.sup.a]    18.17 [+ or -] 0.90
  sea-       2     202.57 [+ or -] 1.02            22.10 [+ or -] 0.57
  weed       3     202.18 [+ or -] 2.14            24.47 [+ or -] 0.73
High ARA     1     196.73 [+ or -] [1.55.sup.ab]   18.34 [+ or -] 1.37
             2     201.98 [+ or -] 1.31            27.28 [+ or -] 1.54
             3     200.34 [+ or -] 1.24            26.08 [+ or -] 0.87
Low ARA      1     204.34 [+ or -] 1.816           18.50 [+ or -] 1.27
             2     207.24 [+ or -] 1.08            18.11 [+ or -] 0.76
             3     201.25 [+ or -] 1.96            26.71 [+ or -] 0.93
Control      1     196.53 [+ or -] [1.50.sup.ab]   17.44 [+ or -] 1.23
             2     202.36 [+ or -] 0.79            21.52 [+ or -] 0.66
             3     203.38 [+ or -] 1.76            24.15 [+ or -] 0.86

                                                          Egg
           Spaw-                                        diameter
Diet       ning             Jelly coat                 ([micro]m)

Red          1     117.53 [+ or -] [3.89.sup.c]    212.52 [+ or -] 0.90
  sea-       2     113.40 [+ or -] 1.37            219.67 [+ or -] 1.00
  weed       3     128.80 [+ or -] 7.77            224.49 [+ or -] 2.27
High ARA     1     103.92 [+ or -] [3.78.sup.cd]   215.06 [+ or -] 1.36
             2     123.51 [+ or -] 3.16            224.88 [+ or -] 2.03
             3     118.08 [+ or -] 4.25            226.61 [+ or -] 1.77
Low ARA      1      92.39 [+ or -] [4.3.sup.d]     218.52 [+ or -] 1.27
             2     115.10 [+ or -] 1.35            231.28 [+ or -] 1.55
             3     117.44 [+ or -] 4.58            227.96 [+ or -] 1.52
Control      1     103.68 [+ or -] [3.84.sup.cd]   213.98 [+ or -] 1.23
             2     121.26 [+ or -] 0.96            222.27 [+ or -] 1.29
             3     117.73 [+ or -] 9.70            227.10 [+ or -] 2.05

                    Ratio of           Percent
                   cytoplasm          ccurrence
           Spaw-     to egg           of primary
Diet       ning     diameter           eggs (%)

Red          1       0.92         0.18 [+ or -] 0.13
  sea-       2       0.92         1.39 [+ or -] 1.39
  weed       3       0.90         2.10 [+ or -] 0.90
High ARA     1       0.92         3.63 [+ or -] 1.23
             2       0.90         0.53 [+ or -] 0.53
             3       0.88         2.55 [+ or -] 1.30
Low ARA      1       0.94         5.55 [+ or -] 2.88
             2       0.90         5.65 [+ or -] 3.10
             3       0.88         1.49 [+ or -] 0.65
Control      1       0.92         3.90 [+ or -] 1.90
             2       0.91         3.98 [+ or -] 2.36
             3       0.90         3.05 [+ or -] 1.25

* Means, within a column, with different superscript letters are
significantly different (p < 0.05).

TABLE 3.

Influence of diet and spawning round on shell length, weight and
weight change of Haliotis laevigata. (Data are means [+ or -] SE)

                                   Shell
              Spawning            length
Diet            round              (mm)

Red seaweed   spawn out    130.28 [+ or -] 1.31
                  1        129.78 [+ or -] 1.27
                  2        129.39 [+ or -] 1.25
                  3        129.89 [+ or -] 1.23
High ARA      spawn out    133.89 [+ or -] 1.37
                  1        133.44 [+ or -] 1.35
                  2        133.47 [+ or -] 1.44
                  3        133.69 [+ or -] 1.45
Low ARA       spawn out    132.39 [+ or -] 1.40
                  1        131.97 [+ or -] 1.39
                  2        131.58 [+ or -] 1.38
                  3        131.62 [+ or -] 1.52
Control       spawn out    133.33 [+ or -] 1.28
                  1        133.31 [+ or -] 1.10
                  2        133.17 [+ or -] 1.14
                  3        133.63 [+ or -] 1.15

              Spawning               Weight
Diet            round                  (g)

Red seaweed   spawn out   336.87 [+ or -] [12.00.sup.a]
                  1       328.50 [+ or -] [10.74.sup.b]
                  2       323.21 [+ or -] [10.95.sup.b]
                  3       323.03 [+ or -] [9.97.sup.b]
High ARA      spawn out   380.24 [+ or -] [16.11.sup.a]
                  1       363.47 [+ or -] [16.24.sup.b]
                  2       360.98 [+ or -] [16.19.sup.b]
                  3       352.57 [+ or -] [14.98.sup.b]
Low ARA       spawn out   352.26 [+ or -] [13.75.sup.a]
                  1       330.18 [+ or -] [14.94.sup.b]
                  2       331.53 [+ or -] [14.65.sup.b]
                  3       327.36 [+ or -] [14.69.sup.b]
Control       spawn out   360.77 [+ or -] [11.92.sup.a]
                  1       348.08 [+ or -] [10.03.sup.b]
                  2       355.55 [+ or -] [10.73.sup.b]
                  3       350.53 [+ or -] [10.61.sup.b]

                           Weight
              Spawning     change
Diet            round       (g)

Red seaweed   spawn out
                  1        -8.36
                  2        -5.29
                  3        -0.18
High ARA      spawn out
                  1       -16.77
                  2        -2.5
                  3        -8.41
Low ARA       spawn out
                  1       -22.09
                  2        +1.35
                  3        -4.17
Control       spawn out
                  1       -12.69
                  2        +7.46
                  3        -5.02

* Means, within a column, with different superscript letters are
significantly different (p < 0.05).

TABLE 4.

Descriptive statistics of egg size frequency distributions spawned by
1-3 female abalone, Haliotis laevigata, per diet treatment over three
consecutive spawning rounds.

                                                  Female

 Treatment    Spawning      Quartile        1        2        3

Red seaweed      1       1. Quartile      218.29   213.69   213.03
                         3. Quartile      223.55   220.92   218.29
                         Interquartile      5.26     7.23     5.26
                         Min ([Lm)        207.77   210.40   210.40
                         Max (Rm)         228.81   223.55   228.81
                 2       1. Quartile      213.69   215.66   218.29
                         3. Quartile      220.92   218.29   223.55
                         Interquartile      7.23     2.63     5.26
                         Min ([micro]m)   205.14   210.40   210.40
                         Max ([micro]m)   223.55   230.13   226.18
                 3       1. Quartile      218.29   213.36   218.29
                         3. Quartile      223.55   219.28   223.55
                         Interquartile      5.26     5.92     5.26
                         Min ([micro]m)   210.40   210.40   210.40
                         Max ([micro]m)   231.44   231.44   230.13
High ARA         1       1. Quartile      210.40
                         3. Quartile      215.66
                         Interquartile      5.26
                         Min ([micro]m)   202.51
                         Max ([micro]m)   220.92
                 2       1. Quartile      213.03
                         3. Quartile      218.29
                         Interquartile      5.26
                         Min ([micro]m)   210.40
                         Max ([micro]m)   230.13
                 3       1. Quartile      218.29
                         3. Quartile      223.55
                         Interquartile      5.26
                         Min ([micro]m)   210.40
                         Max ([micro]m)   228.81
Low ARA          1       1. Quartile      207.77   221.58
                         3. Quartile      210.40   228.81
                         Interquartile      2.63     7.23
                         Min ([micro]m)   199.88   210.40
                         Max ([micro]m)   218.29   236.70
                 2       1. Quartile      220.92   223.55
                         3. Quartile      236.70   231.44
                         Interquartile     15.78     7.89
                         Min ([micro]m)   202.51   218.29
                         Max ([micro]m)   252.48   239.33
                 3       1. Quartile      216.98
                         3. Quartile      223.55
                         Interquartile      6.57
                         Min ([micro]m)   210.40
                         Max ([micro]m)   236.70
Control          1       l. Quartile      212.04   202.18   213.03
                         3. Quartile      218.29   207.77   220.59
                         Interquartile      6.25     5.59     7.56
                         Min ([micro]m)   203.83   194.62   210.40
                         Max ([micro]m)   227.50   216.98   226.18
                 2       1. Quartile      213.03   220.92   210.40
                         3. Quartile      218.29   228.81   215.66
                         Interquartile      5.26     7.89     5.26
                         Min ([micro]m)   205.14   194.62   202.51
                         Max ([micro]m)   226.18   244.59   223.55
                 3       1. Quartile      211.72   216.98
                         3. Quartile      223.55   224.54
                         Interquartile     11.84     7.56
                         Min ([micro]m)   186.73   193.31
                         Max ([micro]m)   243.28   240.65