Effects of season, temperature control, broodstock conditioning period and handling on incidence of controlled and uncontrolled spawning of greenlip abalone (Haliotis laevigata donovan) in western Australia.

ABSTRACT Managing the broodstock conditioning process is important to the successful establishment of an abalone industry that can reliably produce juveniles from captive stock and pursue selective breeding. Four conditioning periods of 6, 8, 10 and 12 wk intervals were tested with captive greenlip abalone (Haliotis laevigata), using a constant conditioning temperature and ambient temperature as a control. The conditioning periods of 6, 8, 10 and 12 wks are equivalent to ~420, 560, 700 and 840 EAT (effective accumulative temperature), based on a biological zero point (BZP) of 6.9[degrees]C for gonad development. Greenlip abalone broodstock collected from the wild (87.5-142.1 mm in shell length and 108.2-482.8 g in whole weight) were spawned in all seasons including numerous induced spawnings outside of the normal breeding period with conditioned stock. Over the whole trial period, greater egg production from prescheduled spawnings occurred in the conditioned group with an average of 1.70 x [10.sup.6] eggs per tub of 5 female abalone per planned spawning, compared with abalone held in ambient control tubs that only produced an average of 0.40 x [10.sup.6] eggs per tub. Animals that were spawned every 8 wks produced the largest average number of eggs per holding tub, however, there was very little difference between conditioning periods in terms of number of successful spawnings per spawning round. The egg production for all planned spawnings was highest before the "natural spawning season" for greenlip abalone. In comparison, the highest numbers of unplanned spawnings occurred around the natural spawning period for the conditioned and control groups. Handling abalone did reduce egg production within the trial, but this was not statistically significant. Histological examination showed that using the visual gonad index is not a good indication of maturation of the abalone and confirmed that abalone can be conditioned out of season.

KEY WORDS: Haliotis laevigata, broodstock conditioning, abalone, handling, conditioning periods, gonad histology.

INTRODUCTION

Aquaculture of abalone is practiced in many countries worldwide and in Australia is an emerging industry across the southern mainland states and Tasmania (Fleming 2000). However, little research has been carried out in Australia on conditioning of mature abalone for use as broodstock. Understanding the broodstock conditioning process is important to the successful establishment of this industry by ensuring reliable and cost effective production of juveniles and for selective breeding (Li 2001). The ability to have ripe individuals year-around or at predictable times in the year allows the culturist to schedule the hatchery process to occur at the most favorable time of the year for growth and survival (Hahn 1989). The use of conditioned abalone overcomes problems relating to the use of wild-caught adult abalone for broodstock including access to stock (through state regulations), variability in the condition of stock and the reliability of successful spawnings (Fleming 2001).

The major species being farmed in Western Australia is the greenlip abalone Haliotis laevigata Donovan. This species is distributed along the southern coastline from western Victoria to Cape Naturaliste in Western Australia and around islands near Tasmania (Freeman 2001). Broodstock conditioning research has been conducted on this species in that state (Daume & Ryan 2004) and on the genetically distinct population (Elliot et al. 2001) in Tasmania, where ambient temperatures are much lower than in Western Australia and exhibit greater seasonal variation (Lleonart 1992, Grubert & Ritar 2002, 2003, Freeman 2001). One of the major problems in investigating abalone reproduction is the short period of natural spawning (Hahn 1989). The natural spawning season for greenlip abalone on the south coast of Western Australia is from late September/early October through to December (Wells & Mulvay 1992).

Several biological processes are involved when conditioning abalone, for example, gametogenesis for females and males, synthesis of hormones controlling gametogenesis and spawning, and for the female only, vitellogenesis and synthesis of hormones controlling vitellogenesis. In addition to the gonad being ripe, the animal must be in the proper physiological state and in appropriate environmental conditions to promote spawning (Hahn 1989). Many factors have been found to influence conditioning in different species of abalone including water temperature, food, photoperiod or a combination of these variables (Hahn 1989). Uki and Kikuchi (1982) considered nutrition as an important factor for gonad development in abalone and Webber (1970) found that water temperature controls the reproductive cycle for many marine gastropods. In most species of abalone, temperature strongly influences rates of gonad development; its effect is cumulative above a certain threshold temperature and is known to vary between species (Grubert & Ritar 2003). The threshold temperature, better referred to as the "biological zero point" (BZP) was first identified by Kikuchi and Uki (1974). They determined the effective accumulative temperature in degree-days (EAT[degrees]C-days) for gonad conditioning of Haliotis discus hannai Ino and Haliotis discus Reeve by subtracting the BZP from the daily water temperature and adding this figure over the conditioning period (in days). H. discus hannai was found to have a BZP of 7.6[degrees]C (Uki & Kikuchi 1984). Grubert and Ritar (2003) studied the effect of temperature and conditioning interval on the spawning success of wild-caught blacklip (Haliotis rubra Leach) and greenlip abalone fed an artificial diet. They found that unlike 14. rubra, male and female H. laevigata have a higher spawning rate and gamete production when conditioned at 16[degrees]C compared with 18[degrees]C. In a preceding experiment, Grubert and Ritar (2002) estimated BZP values for gonadal and larval development for H. rubra as 7.8[degrees]C and 7.9[degrees]C respectively and for H. laevigata 6.9[degrees]C and 7.2[degrees]C respectively. In Lleonart's (1992) research into broodstock conditioning of greenlip abalone, the mean temperature during conditioning was 16[degrees]C and the number of elapsed degree-days was recorded as 1,750, which when corrected using a BZP of 6.9[degrees]C gives an EAT of 995 degree days. After this time, the abalone were only just coming into condition. For this study on broodstock conditioning in greenlip abalone, a BZP of 6.9[degrees]C was assumed.

The aim of this study is to develop a sound protocol for conditioning broodstock abalone for commercial purposes by determining the most favorable conditioning period (e.g., 6, 8, 10 or 12 wks) as indicated by a visual condition index, histological assessment, spawning success rate (as a % of the group subjected to spawning induction), fertilization rate and hatch rate. Seasonal patterns for controlled and uncontrolled ("wildcat") spawnings and whether handling stress influences the success of broodstock conditioning and initial spawning success were also investigated. Gonad histology and macroscopic observation of the gonad were assessed as relative guides to the likelihood of spawning success.

METHODS

The term "conditioning" is used here for the process adopted to induce gonad ripeness and ability to spawn in captive adult abalone, usually outside of the normal spawning season but also to allow multiple spawnings within that season.

Experimental Set-up

The research was conducted at a commercial hatchery, Great Southern Marine Hatcheries (GSMH), Albany (117[degrees]57'38"E, 35[degrees]5'52"S), Western Australia. Wild abalone were collected from Bremer Bay, approximately 200 km east of Albany, and held in the conditioning system for up to 7 mo before the trial commenced, where broodstock were acclimatized and temperature control was refined. The trial ran for 36 wks from April 2001 to December 2001. The culture system consisted of 60, 30-L round plastic tubs, with 8 male tubs and 24 female tubs at 17.0[degrees]C [+ or -] 0.4[degrees]C in the conditioning room, and 8 male and 8 female control tubs in an outdoor grow-out area to provide ambient conditions (e.g., natural photoperiod and temperature with unfiltered sea-water). The remaining 14 tubs of females were used for histology and were also held in the conditioning room at 17.0[degrees]C [+ or -] 0.4[degrees]C. Each tub contained 5 abalone and males and females were held in separate tubs. The abalone used in this experiment were of mean shell length 118.1 mm mean size (range 87.5-142.1 mm total shell length) and 250.4 g whole weight (108.2-482.8 g).

The continuously aerated tubs had a flow rate of 1-1.5 L/min ([??] 2-3 water exchanges per hour) and a central standpipe. Water supplied to the reservoir (5,000 L) and heater/chiller unit was filtered down to 10 [micro]m using a bag filter. A photoperiod of 12 h low light and 12 h darkness was used for animals that were kept indoors.

Spawning Groups

All of the tubs except those set aside for histological examination were randomly assigned into 4 groups (6, 8, 10 and 12 wk spawning intervals). This is equivalent to ~420, 560, 700 and 840 EAT based on a BZP of 6.9[degrees]C. Over a 36-wk period, groups were spawned at 6, 8, 10 and 12 wk intervals respectively (Table 1). Each spawning group consisted of 6 female tubs and 2 male tubs from the conditioning room and 2 female tubs and 2 male tubs under ambient conditions (controls).

All animals were induced to spawn following commercial spawning protocols without being removed from their holding tubs. This strategy was aimed at minimizing handling of the abalone. Therefore for all spawnings, egg production was recorded per tub of 5 female abalone not per individual female. Animals from the histological examination group were not deliberately induced to spawn during the trial. Two tubs of females from this group were sacrificed every 4 wk for analysis by histology (see below).

Spawning success (%) was calculated for each planned spawning by:

Number of tubs of abalone that spawned x 100 / Total number of tubs of abalone that were subjected to spawning induction stimuli

Unplanned ("wildcat") spawnings also took place. As with data for planned spawnings in which the number of spawning inductions depended on the conditioning interval (6-12 wk), estimates of incidences of spawning and egg production had to take into account the number of tubs for each treatment (conditioned or control). Similarly data analysis by season also was corrected for the number of days of each season (Spring, Summer, Autumn, Winter) encompassed within the 36-wk trial.

Wildcat spawnings (%) for each month was calculated by:

Number of tub x day combinations per month with evidence of wildcat spawnings x 100 / Number of days in month x Number of tubs for control (= 16) or conditioned (= 32)

Similarly, wildcat spawnings (%) per season for each conditioning period and controls was calculated by:

Number of tub x day combinations per month with evidence of wildcat spawnings x 100 + No of days in season x Number of tubs for conditioning group (= 8) or controls (= 4)

Feeding and Cleaning

All individuals were fed daily ad libitum with a commercial formulated, extruded conditioning diet (Adam and Amos Feeds, South Australia). Feed was given as a rate of 1% to 2% body weight per day.

Each tub of abalone were cleaned daily (morning) with as little disturbance as possible. Complete drainage of each tub occurred three times a week. Every other day, tubs were just siphoned clean to minimize stress related to cleaning procedures. Rinsing water for the tubs was maintained at the same temperature as the culture vessels to eliminate possible spawnings from temperature shock.

Histology

Histological assessment was carried out on female greenlip abalone to determine progress with conditioning. Egg development was assessed every 4 wks, over a period of 24 wks, commencing April 11, 2001 by sacrificing two tubs of female abalone from the histological examination group and fixing them whole in 10% neutral buffered formalin. Gonad samples from the fixed samples for histology were cut from the abalone, dehydrated, embedded in paraffin wax, sectioned at 5 [micro]m and stained with hematoxylin and eosin using standard techniques. The gonads were examined and classified using the stages described in the abalone atlas produced by Handlinger et al. (2001). The stages they describe are 1 = Recovery phase gonads; 2 = Growing phase gonads; 3 = Mature ovaries; 4 = Atretic ovaries and 5 = Resting phase ovaries.

Handling to Assess Gonad Index

Throughout this experiment, abalone in the conditioning room, except those used for visual gonad index (below) and histology assessment, were never removed from their tubs. These tubs were used as the nonhandled control group for the assessment on handling versus nonhandling.

Every 14 days after the start of the experiment, two female tubs from each spawning group (6, 8, 10, 12 wk) were weighed, measured and visually assessed to determine the level of gonad ripeness. The same two tubs of animals were chosen each time to assess gonad ripeness, growth data over time and for the effects of handling. Abalone were removed by lifting them off the bottom with a spatula-like tool. The progression of gonad development was determined for each individual using a visual scoring system of 0-3 and was referred to as the visual Gonad Index (GI). A score of 0 = cannot determine the sex, 1 = the upper edge of the gonad was below the rim of the shell, 2 = the upper edge of the gonad was even with the rim of the shell, and 3 = the upper edge of the gonad was above the rim of the shell. This means that at a score of 1 the sex of the abalone can be determined, at a score of 2 there is a possibility the abalone may spawn and at a score of 3 it is assumed the abalone is highly likely to spawn. The egg production results were used to assess the influence of handling on spawning success, in comparison with the remaining 4 tubs of females from the conditioning room for each spawning group.

Experiment Initiation

All broodstock abalone used in the experiment were tagged and initial weight and shell length data were recorded. To initiate the experiment commercial spawning methods were used to induce spawning. All abalone were exposed to 18.5[degrees]C water temperature for 12 h, then desiccated for 45 min followed by an ultra violet (UV) treated water temperature shock of 21[degrees]C, which was slowly decreased back to 17[degrees]C over a period of 8 h. Animals were held in the UV treated water for a maximum of 24 h to induce spawning. After this time they were returned to their normal experimental conditions in the conditioning room or control area. The GI for each abalone were assessed and recorded during the desiccation time.

Data Recording

A temperature data logger was placed into the reservoir. Daily temperatures were also recorded manually. Water quality was checked weekly (DO, pH, ammonia and salinity). Atmospheric pressure readings were recorded on a daily basis.

Throughout the experiment any spawnings that occurred were recorded, including "wildcat" spawnings, which are spawnings that occurred unexpectedly throughout the trial. All results using data from "wildcat" spawning events excludes "wildcat' spawning data from the histology tubs. The natural spawning season for greenlip abalone was considered to be September to December. Where possible, numbers of eggs per tub and per group were recorded. Total number of eggs per batch was estimated from 3 sub-samples as:

Average number of eggs in 1-mL samples x Total volume of the sample.

Data Analyses

Statistical analyses were carried out using the STATISTICA 6.1 (Stat Soft, Inc. 2002) computer package. The assumptions of normality and homogeneity of variance were confirmed graphically for each data set using box plots. Egg production data between conditioned and control tubs, and handled and non-handled tubs were compared with t-tests. Spawning success was also compared with t-tests between males and females. Spawning success, egg production and histological egg stages between conditioning intervals were analyzed using l-way ANOVA. Relationships between the wildcat spawning and daily barometric pressure were explored using simple regression analyses.

RESULTS

Water Temperature and Quality

Water temperatures in the conditioning room were usually constant between 16.6[degrees]C to 17.2[degrees]C, whereas temperatures in the control tubs ranged from 14.0[degrees]C to 20.2[degrees]C (Fig. 1). Dissolved Oxygen ranged from 76.9% to 105.0% (mean 90.0%); pH was within the range of 7.78-8.29 (mean 8.17), whereas salinity was recorded between 35.2 ppt and 36.0 ppt with a mean of 35.5 ppt. Free ammonia concentrations were less than 0.1 mg N[H.sub.3]-N/1.

[FIGURE 1 OMITTED]

Survival and Growth

Excluding abalone for histological assessment, high survival (>97%) was observed throughout the trial for all greenlip abalone held in the conditioning room and control area.

Mean average shell length for the conditioned and control tubs of abalone over the whole trial period of 36 wks was calculated at 6.03 [micro]m/day and 5.07 [micro]m/day respectively, whereas the mean average weight gain per abalone for the conditioned and control tubs of abalone, were 30.80 [micro]g/day and 31.82 [micro]g/day respectively.

Spawning

In initiating the experiment no abalone actually spawned. However, wildcat spawnings (unplanned spawnings) had occurred in the majority of tubs within the 2 wks before initiating the experiment.

Wildcat spawning data for abalone held in the control groups showed that in April, outside of the natural spawning season, there were some wildcat spawning events occurring, whereas in May through to August, again outside the natural spawning season, there were very few wildcat spawning events (Fig. 2). However, in September through to December, during the natural spawning season, many more wildcat spawnings were recorded in both conditioned and control animals. For the planned spawning groups (every 6, 8, 10 and 12 wks), all wildcat spawning data showed similar patterns (Fig. 3). However, it was evident that more wildcat spawning events occurred in summer, within the natural spawning season in the controls (Fig. 3) despite the trial covering only 1 month in summer.

[FIGURES 2-3 OMITTED]

Planned spawning results showed that, over the whole trial period, egg production in the conditioned abalone was significantly greater than egg production in the controls (df = 28, t = 2.299, P = 0.029). Conditioned abalone produced 1.70 million eggs per female tub per planned spawning compared with abalone held in the control tubs that only produced an average of 0.40 million eggs per female tub (Fig. 4). Highest egg production was recorded within the winter months, outside the natural spawning season for abalone held in the conditioning room with an average of 3.16 million eggs per tub of 5 female abalone per planned spawning (Fig. 5). In comparison, for abalone held in the control tubs, the highest average egg production of 0.87 million eggs per tub of female abalone was recorded during spring (Fig. 5). Egg production from planned spawnings was lowest from abalone held in the conditioning room during the summer months (Fig. 5).

[FIGURES 4-5 OMITTED]

Results for the spawning intervals of 6-, 8-, 10- and 12-wk periods showed that abalone from the week 8 spawning interval had the highest egg production during the second spawning induction (week 16) of 44.23 million per tub of female abalone (Fig. 6). Egg production was very low in the first planned spawning attempt for spawning intervals of 6 and 8 wk of 2.5 million and 1 million respectively. However, spawning occurred in only about 13% of female tubs of abalone spawned during both inductions. For spawning intervals of 10 and 12 wks the opposite was observed, with highest egg production of 23 million and 19 million occurring within their first planned spawning attempt and a decline in egg production for the following planned spawning attempts (Fig. 6). During the first planned spawnings, the percentage of female tubs in which abalone spawned were 75% and 38% for spawning intervals 10 and 12 wks respectively. For the latter inductions for these spawning intervals, where egg production declined, the percentage of actual spawnings also declined to 38% and 25% in both spawning intervals for the second and third spawning attempts.

[FIGURE 6 OMITTED]

Overall, the 6 wk spawning interval had the highest average percentage spawning success of 60%, whereas the 12-wk spawning interval had the lowest of 33% (Fig. 7). However, there was no significant difference between spawning intervals for spawning success (df = 3, F = 0.164, P = 0.917).

[FIGURE 7 OMITTED]

Average numbers of eggs produced for each interval showed week 8 to be slightly higher than intervals of 6, 10 and 12 wks with an average egg production of 12 million eggs produced per tub of 5 female abalone (Fig. 8). However, overall there was no significant difference between spawning intervals in terms of eggs produced per tub of female abalone (df = 3, F = 0.150, P = 0.928).

[FIGURE 8 OMITTED]

There was no significant difference (t = 1.18, df = 38, P = 0.25) between the percentages of males and females that spawned in each planned spawning induction.

No relationship was observed between wildcat spawning events and average daily barometric pressure (r = 0.06) or between wildcat spawning events and change in daily pressure (r = 0.01).

Fertilization and hatch rates were consistent between each spawning group throughout the trial. In general, all lines produced from conditioned and control stock displayed very high fertilization (90% to 100%) and hatch rates (80% to 100%).

Histology

Egg stages expressed as percentage of mature (stage 3) gonads from the histological examination results were not comparable to the results obtained for GI (Table 2). However, egg stages over time showed a significant difference (df = 6, f = 4.654, P < 0.001). A significant drop was observed for the average egg stage between 0 and 4 wks (P = 0.005), whereas a significant increase for the average egg stage was evident between 4 and 16 wks (P = 0.019) as well as between 4 and 20 wks (P < 0.001).

Handling

Overall averages for egg production for the whole trial showed that nonhandled abalone produced more eggs (2.06 [+ or -] 0.68 million per tub of female abalone per planned spawning) than abalone from the handled group (0.98 [+ or -] 0.29 million) (Fig. 9). However, this difference was not significant (df = 28, t = 1.459, P = 0.156). Percentage spawning success rates for nonhandled and handled abalone were very similar and not significantly different, with values of 55% and 57% (df = 28, t = 0.14, P = 0.889).

[FIGURE 9 OMITTED]

DISCUSSION

Water Temperature and Quality

The broodstock conditioning system used provided stable water temperature and high water quality. In Albany, ambient water temperatures exhibit a smaller range and reach much warmer temperatures than in Tasmanian waters (Hindrum et al. 1996). At warmer temperatures it is evident that abalone grow faster (Freeman et al. 2000).

In the conditioning room a water temperature increase to 19.1[degrees]C in mid April and a decrease to 14.7[degrees]C in early September can be observed in Figure 1. These fluctuations corresponded to power failures to the heater/chiller unit.

Survival and Growth

All broodstock abalone showed high survival (97%) but low growth rates between the start and finish of the trial. There was very little difference between mean average weight gain and mean average shell length for both the control and conditioned tubs of abalone. The overall averages of 5.77 [micro]m/day for average shell length and 31.54 mg/day for average weight gain for broodstock abalone are slow when compared with growth rate estimates, used for stock assessment purposes, for wild adult greenlip abalone of 25 mm/year in southern Western Australia (A. Hart pets. comm.). Moreover, they are low when compared with growth rates observed for wild juvenile greenlip abalone of 1,690 [micro]m/month (Shepherd 1988). Shepherd and Hearn (1983) believe that energy expenditure during gonad development can reduce growth rate. Growth rate is affected by several variables including genotype (Brown 1991), density, type and amount of feed (Day & Fleming 1992), water flow (Higham et al. 1998), water quality (Harris et al. 1998) and handling techniques such as frequency of emersion (Maguire et al. 1996).

Spawning

Within the 2 wks prior to the start of this experiment, wildcat spawning occurred in the majority of tubs of abalone. This could explain why we did not get any induced spawnings when all tubs of abalone were subjected to spawning stimuli at the beginning of the experiment.

Wildcat spawning data for abalone held in the control groups followed the general pattern of a natural spawning cycle for greenlip abalone. In the warm month of April there were some wildcat spawning events occurring, but in the cooler months of May to August there were very few wildcat spawning events. However, in the warmer months of September to December in the natural spawning season for greenlip abalone a lot more wildcat spawnings were evident (Fig. 2). Shepherd and Laws (1974) found the spawning season for greenlip abalone in South Australia to be October to March, whereas Wells and Mulvay (1992) concluded that it was September to December in Western Australia. The results presented in this study show that greenlip abalone can be spawned outside of the natural spawning season. The egg production of conditioned broodstock from planned spawnings was highest before the natural spawning season commenced (Fig. 5). Fewer successful planned spawning events and lower egg production occurred around the natural spawning season. However, a high number of wildcat spawning events occurred in all tubs during this period. Low egg production would be expected per tub of female abalone during the planned spawnings over the natural spawning season caused by abalone already expelling their eggs in unplanned spawning events.

Spawning events before the natural spawning season can be highly beneficial to farmers because they can take advantage of the enhanced growth of juveniles during the early summer months. Animals can be weaned off the plates before the highest summer temperatures occur. High water temperature during the weaning process can cause high mortalities in some regions of Southern Australia (M. Russell pers. comm.).

The planned spawning results showed that over the whole trial period the conditioned group of abalone produced around four times as many eggs as the control groups. This could partly be explained for 2 reasons. Firstly, the control groups did not produce many eggs in winter and secondly, a lot of egg production for the control group was "wasted" on wildcat spawnings (where egg production is not recorded). However, it still strongly suggests that holding broodstock abalone in a conditioning system has a major advantage over holding broodstock at ambient conditions. In recent years, most Australian farmers have held their broodstock in ambient conditions on their farm and only increased feeding and feed type to condition their broodstock. Many studies have suggested that holding abalone at constant temperature and feeding a good quality diet will condition broodstock abalone (Kabir 2001, Grubert & Ritar 2003, Lleonart 1992, Moss 1998).

It was observed that the week 8 spawning interval produced a larger number of eggs per tub of abalone compared with all other spawning week intervals. The data for the first planned spawning for the week 8 spawning interval shows low egg production. This could suggest that most abalone in this group for the second planned spawning were actually conditioned for 16 wk and demonstrates the benefits of longer conditioning periods. This trial was only set to test a maximum interval of 12 wks. Further studies into longer conditioning times are now in progress.

Histology

Histological assessment of egg stages in greenlip abalone is more accurate in determining the maturity of the abalone than using the visual GI scoring system.

Histology results show a significant drop between 0 and 4 wks, suggesting that spawning or reabsorption of eggs may have reduced the average egg stage for that period. The significant increase in average egg stage between 4 and 16 wks and 4 and 20 wks suggests that the abalone come into condition around this time, which equates to 12 and 16 wks for conditioning. The need for a longer conditioning period for this species was also evident in Grubert and Ritar (2003), who found for greenlip abalone in Tasmania, egg production peaked when held at 18[degrees]C for 114 days (1265 EAT[degrees]C-days). Assuming the BZP of greenlip abalone is 6.9[degrees]C (Grubert & Ritar 2002) then this equates to approximately 18 wk at our trial temperature of 17[degrees]C. In another study in Tasmania, Leonart's adjusted results of 16 wks at 17[degrees]C, also shows that a longer conditioning period is needed (Lleonart 1992).

In terms of the average number of eggs produced, weeks 6 and 8 were not very successful, which suggests that the abalone are not in condition after such short conditioning periods. Conditioning intervals of 10 and 12 wks appeared to be more successful. The first planned spawning for both the 10 and 12 wks intervals showed good egg production. The second and third planned spawning events for these two groups were not as successful because of high numbers of wildcat spawning events that appear to be uncontrollable in the warmer months of the year. Clearly, greenlip abalone do need at least 10 wks for conditioning.

Generally, most farmers use the GI as an indication of the abalone's spawning ability. However, results from the histological examination showed that visually inspecting the gonad was not a good indication of the actual level of gonad development. There is no evidence that the GI is a good indication of mature abalone eggs (Table 2). More recently, farmers have noticed that a large gonad does not necessarily mean that the abalone will spawn.

Handling

Handling abalone did reduce egg production within the trial but this was not statistically significant. The nonhandled abalone produced approximately twice the average number of eggs than abalone that were handled regularly throughout the trial. This may not have been statistically significant because of the difference in the number of replicates used for the handled and non-handled groups of 2 tubs and 4 tubs respectively. Potentially, there may not have been enough power to detect a significant difference. Hone et al. (1997) found that in the wild abalone near condition will spawn if high stress conditions occur. Abalone farmers have suggested that handling stresses abalone.

There was no difference between the percentages of males and females that spawned in each planned spawning induction. This does not follow the same pattern that was described by Hahn (1989), that in the hatchery when males and females are induced to spawn, a higher percentage of males spawn compared with females. However, our observation that males generally spawned before females confirmed that of Hahn (1989). In this trial, males usually spawned before females and commenced about 11-12 h after induction with UV. Initially, spawnings had finished ~17-18 h after UV induction. However, later observations showed that there were 2 "periods" of spawnings, the first being early to late evening (~11-18 h after UV induction) and the second being around dawn (~24 h after UV induction).

This system is suitable as a commercial conditioning system as it reduces labor costs in handling and provides less stress to the abalone before spawning. However, in comparison it would be less suitable for genetic programs based on single pair crosses because the abalone are held in groups and not removed from the tubs before spawning.

ACKNOWLEDGMENTS

The authors thank the technical staff Gaynor Jones, Ian Cameron and Dianne Watson who maintained and assisted with spawning the abalone, the histology unit at the Department of Agriculture Western Australia for technical assistance in the processing of the histology samples and Dr Brian Jones who undertook the gonad staging from the histological slides.

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KYLIE FREEMAN, (1) * SABINE DAUME, (1) MATTHEW ROWE, (1,2) STEVE PARSONS, (2) RIC LAMBERT (2) AND GREG B. MAGUIRE (1)

(1) Research Division, Department of Fisheries Western Australia, PO Box 20, North Beach WA 6920;

(2) Great Southern Marine Hatcheries, PO Box L34, Little Grove, Albany WA 6330.

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

TABLE 1.

Spawning schedule for each group of abalone.

 Group                  Spawning weeks (from day 0 *)

 6 Week    6 (May)    12 (July)   18 (Aug)    24 (Sept)   30 (Nov)
 8 Week    8 (June)   16 (July)   24 (Sept)   32 (Nov)       --
10 Week   10 (June)   20 (Aug)    30 (Nov)       --          --
12 Week   12 (July)   24 (Sept)   36 (Dec)       --          --

* Day 0 was the day when an attempt was made to spawn out
all abalone after collection.

TABLE 2.

Gonad index (GI) and percentage of mature egg stage (stage 3) for
histology tissue sections averaged for 10 abalone, which were
collected every four weeks over a period of 24 weeks.

             Average           Average % of Mature Stages
              Gonad              (Stage 3) for Each Week
Week         Index *         Histological Samples Were Taken

 0     1.15 [+ or -] 0.15          54.17 [+ or -] 0.10
 4     1.40 [+ or -] 0.16          20.00 [+ or -] 0.10
 8     1.10 [+ or -] 0.07          36.67 [+ or -] 0.12
12     1.10 [+ or -] 0.07          56.67 [+ or -] 0.15
16     1.20 [+ or -] 0.11          36.67 [+ or -] 0.13
20     0.89 [+ or -] 0.11          40.74 [+ or -] 0.13
24     1.00 [+ or -] 0.00          58.33 [+ or -] 0.08

* For gonad index (GI) a score of 0 = cannot determine the sex,
1 = the sex of the abalone can be determined but spawning is unlikely,
2 = there is a possibility the abalone may spawn and 3 = it is assumed
the abalone is highly likely to spawn.