Triploidy induction in Australian greenlip abalone Haliotis laevigata (Donovan) with cytochalasin B

Triploid induction in Australian greenlip abalone, Haliotis laevigata (Donovan), was conducted by blocking the formation of the second polar body using cytochalasin B (CB). Twenty minutes after fertilization, the zygotes of greenlip abalone were treated with four CB concentrations (0, 0.25, 0.5 and 0.75 mg L⁻¹) for 10, 15 and 20 min. The ploidy of resultant larvae was determined using flow cytometry at 72-h post fertilization. Our study showed that fertilization, hatching, survival and induced triploidy of abalone larvae were significantly affected by the CB concentration and treatment duration. The effective range of CB concentration for triploid induction on greenlip abalone was 0.5–0.75 mg L⁻¹ with an induction duration of 10–15 min. The results indicate that the most effective treatment combination for triploid induction in greenlip abalone is 0.5 mg CB L⁻¹ for 15 min starting at 20-min post fertilization.     

Modelling the growth of cultured greenlip abalone (Haliotis laevigata Leach): practical modes of measurement

Commercial abalone growers and aquaculture researchers need to monitor and compare the growth rate of their stocks. Growth rates based on both length and weights are often calculated, but the process of disturbance for measurement will affect the results of most studies. In natural populations, constant (linear) growth in length has been supported, while logarithmic transformations for growth rates (length and weight) have been used for aquaculture studies. This study of growth of greenlip abalone (Haliotis laevigata, Leach) over a prolonged period (3 years) indicates that both data treatments are suitable. If weights are being used, then a growth model that allows for exponential growth, such as specific growth rate (SGR), must be used. If lengths are measured, then the calculation of simple differences in the lengths over time is sufficient. The apparent paradox in use of two forms of measure is comfortably rationalized as a result of the weight–length relationship and the von Bertalanffy relationship for this species. Thus, methods of minimal disturbance and stress, such as photographic estimation of length, could be used to accurately monitor growth trends in aquacultural studies as well as in industry.     

Cryopreservation of sperm in farmed blacklip abalone (Haliotis rubra Leach, 1814)

This study developed a technique of sperm cryopreservation using liquid nitrogen (LN) vapour in farmed blacklip abalone Haliotis rubra through evaluating the following five key factors: (1) cryoprotectant agent (CPA) toxicity; (2) cooling temperature; (3) thawing temperature; (4) sperm to egg ratio and (5) sugar addition, using sperm motility or fertilization rate as quality assessment indicators. The results demonstrated that 6% dimethyl sulfoxide (DMSO) was the best single CPA for sperm cryopreservation in this species. The highest post‐thaw sperm motility was achieved when sperm were exposed to LN vapour for 10 min at 5.2 cm above the LN surface and thawed at 60°C and recovered at 16°C in seawater baths. Post‐thaw sperm motility was found to be significantly higher when 6% DMSO was used in combination with 1% or 2% glucose than 6% DMSO alone. Further evaluation of fertilization rate between these CPAs showed that 6% DMSO+2% glucose achieved the highest fertilization rate of 70% at a sperm to egg ratio of 10 000:1.     

Effects of oxygen supersaturation and temperature on juvenile greenlip, Haliotis laevigata Donovan, and blacklip, Haliotis rubra Leach, abalone

Growth and survival of juvenile greenlip (39.03 (SD 3.80) mm (n=524)) and blacklip (31.92 (SD 4.19) mm (n=531) abalone were investigated at high dissolved oxygen levels (95–120% saturation) between 17 and 19°C. Abalone were fed the same artificial diet and each species was contained in groups of approximately 30 individuals within triplicate tanks for each of six treatments and were exposed to flow through water adjusted to give experimental conditions for up to 75 days. Blacklip abalone held at 16.9°C and 97% oxygen saturation grew in shell length significantly faster than all other treatments of blacklip abalone held at 19°C, and significantly faster than blacklip abalone maintained at 111% oxygen saturation and 17.5°C. Both temperature and oxygen saturation significantly affected the survival of this species. Blacklip abalone held at 19°C had significantly lower survival for both 96% oxygen saturation and 120% oxygen saturation, compared with blacklip abalone maintained at either 110% oxygen saturation and 19°C, or for any 17°C treatment. No significant differences were noted for greenlip abalone within the range tested in terms of growth rate, food consumption rate or survival, indicating that greenlip abalone tolerated these conditions better than did blacklip abalone.     

Digestive protease characterization, localization and adaptation in blacklip abalone (Haliotis rubra Leach)

Digestive protease in blacklip abalone (Haliotis rubra Leach) from whole gut extracts was found to have dual pH optima at pH 3 and 10 and an optimum temperature of 45 °C. Over the biologically relevant range of pH (5–8), protease activity dropped to a minimum at pH 5 (53% of the maximal rate at pH 3) rising gradually and continuously up to (and beyond) pH 8. Over the biologically relevant range of temperatures (9–24 °C), protease activity increased continuously with activity at 24 °C being 75% higher than activity at 9 °C. Protease digestion was relatively uniform in gut extracts from sections containing gut contents. Digestion thus appears to be significantly extracellular (in the lumen of the gut). Analysis of gut sections washed free of food matter suggests the anatomical origin of protease from activity of gut regions in the order: digestive gland » salivary gland ≈ stomach > crop > intestine > upper oesophagus. Whole gut protease levels did not alter in response to a high protein artificial diet.     

Optimum temperature for growth of Australian abalone: preferred temperature and critical thermal maximum for blacklip abalone, Haliotis rubra (Leach), and greenlip abalone, Haliotis laevigata (Leach)

The preferred temperature and critical thermal maximum of Australian blacklip abalone, Haliotis rubra (Leach), and greenlip abalone, Haliotis laevigata (Leach), were found to differ only slightly; the blacklip abalone exhibited lower temperature tolerance and preference, as expected from its habitat distribution. Preferred temperatures were 16.9 and 18.9°C, and 50% critical thermal maxima were 26.9 and 2 7.5°C for blacklip and greenlip abalone, respectively. The optimum temperatures for growth calculated from each of these indices and averaged were 17.0 and 18.3°C, respectively.     

Growth and reproductive performance of triploid and diploid blacklip abalone, Haliotis rubra (Leach, 1814)

Growth and reproduction of triploid and diploid blacklip abalone Haliotis rubra (Leach, 1814) were compared in a 30-month study. Triploidy was induced by inhibition of the second polar body formation using 6-dimethylaminopurine (6-DMAP) or cytochalasin B (CB). There were no significant differences in growth and survivorship between triploid and diploid abalone. However, triploid abalone had a more elongated shell and greater foot muscles than diploid abalone. A slightly curvilinear growth in shell length was conformed to all treatments. While diploid abalone had reached sexual maturity and spawned during the study, gonadal development and gamete maturation were abnormal in triploids. Female triploids lacked an apparent gonad at the macroscopic level but microscopic examination revealed that they had a thin layer of oogonia development. In contrast, male triploids were able to form similar-sized gonads to diploids during most of the reproductive period, but with brown-yellow discolouration and stalled gametogenesis at spermatocyte formation. Sex ratio of triploid abalone did not deviate from 1:1. With the onset of sexual maturation, growth and gonadal maturation occurred concurrently in diploid abalone, and there was no indication that growth of (diploid) abalone was reduced.     

The effect of temperature and conditioning interval on the spawning success of wild-caught blacklip (Haliotis rubra, Leach 1814) and greenlip (H. laevigata, Donovan 1808) abalone

Wild‐caught blacklip (Haliotis rubra, Leach 1814) and greenlip (H. laevigata, Donovan 1808) abalone fed a formulated feed were held at 16 or 18°C for different conditioning intervals ranging from 114 to 235 days and induced to spawn using ultraviolet‐irradiated seawater. They were conditioned again for a second identical period before another induction. For H. rubra, mean spawning rate of both sexes was higher in groups held at 18°C than at 16°C, as was the repeat spawning rate. Conversely, animals held at 16°C produced significantly more gametes than those at 18°C. Egg production peaked in groups held at 16°C for ≥165 days. While both mean and total sperm production of H. rubra varied significantly, both figures were always high. Unlike H. rubra, the spawning rate, repeat spawning rate and gamete production of both sexes of H. laevigata were higher when cultured at 16°C than at 18°C. Egg production peaked in groups conditioned at 16°C for ≥212 days. Both mean and total sperm production by H. laevigata were much lower than for H. rubra. This study demonstrates that year‐round hatchery production of seedstock of both species is possible providing broodstock are held under favourable environmental conditions, preferably 16°C.     

Optimization of triploidy induction in the blacklip abalone, Haliotis rubra (Leach, 1814), using 6-dimethylaminopurine

Optimal conditions of 6‐dimethylaminopurine (6‐DMAP) for triploidy induction in the blacklip abalone Haliotis rubra (Leach, 1814) were investigated, targeting inhibition of second polar body (PB2) formation. Two experiments were conducted at a water temperature of 17.5–18.5°C where factorial combination of (1) four dosages (DSs) of 100, 150, 200 and 250 μM 6‐DMAP, four starting times (STs) of 15, 20, 25 and 30 min post fertilization, and two treatment durations (TDs) of 20 and 30 min and (2) three DSs of 50, 100 and 150 μM 6‐DMAP, three STs of 15, 20 and 25 min post fertilization, and three TDs of 10, 20 and 30 min, were applied respectively. Day 3 larvae were sampled for triploidy and survival. Percent triploidy was verified using flow cytometry (FCM). Results show that optimal inductions that combine both high rates of triploidy and reasonable survival were those treatments commenced 15 or 20 min post fertilization and continued for 20 or 30 min, using 100 μM 6‐DMAP. These conditions induced rates of triploidy and relative survival of 80.5–93.3% and 36.5–40.2%, respectively, in the first experiment, and corresponding rates were 79.1–93.6% and 20.7–43.0% in the second experiment. High percent triploidy were also obtained in a number of treatments using 150 μM 6‐DMAP, but with overall survival rates generally lower than those using 100 μM 6‐DMAP.     

Supplementary oxygen and temperature management during live transportation of greenlip abalone, Haliotis laevigata (Donovan, 1808)

Live greenlip abalone, Haliotis laevigata , are highly valued in Australian export markets with demand increasingly being met with cultured stock. Live transportation of abalone requires the maintenance of favourable conditions within transport containers for periods exceeding 35 h. We examined the combined effects of temperature regulation (ice provision) and of supplemental oxygen (60% and 100% concentrations) on mortality rates of abalone over 7 days following a 35-h simulated live-transport experiment. We also examined the physiological condition of greenlip abalone (oxygen consumption rate, haemolymph pH and weight) during the simulation experiment. The provision of ice and supplementary oxygen reduced abalone mortalities. Omission of ice and supplementary oxygen during the transport simulation resulted in mortality rates ranging from 70% to 100%. The addition of ice to containers with ambient oxygen concentrations decreased average mortality rates by 50%. While supplementary oxygen further reduced these rates, the provision of both ice and 100% oxygen was by far the most effective combination, reducing mortalities to between 2% and 6%. Supplementary oxygen increased oxygen consumption rates of abalone above those transported at ambient oxygen concentrations. Live-transport decreased haemolymph pH in all treatments but was most pronounced in treatments without ice or supplementary oxygen. On average, abalone lost 7–13% of their weight during the simulation but this loss was independent of transport treatment.     

A simple model for the determination of the relative utilization efficiency of protein by blacklip abalone (Haliotis rubra Leach)

A 6-week growth rate trial of 6.7 mm ± 1.35 SD long blacklip abalone ( Haliotis rubra ) fed one of four graded protein feeds (26%, 32%, 37%, 45% crude protein, CP) resulted in a maximum average growth rate of 81 μm day⁻¹ (percentage daily weight gain of 5.2% day⁻¹) with the 45% CP diet. Based on the results, a new, simple, non-destructive method to calculate the relative utilization efficiencies (RUE) of nutrients is presented to estimate the relative contributions of a nutrient to growth, body maintenance and the excess used for energy. Calculations of RUE of protein estimated that at the maximum growth rate, 59% of the CP was responsible for growth, while 31% was used for body maintenance and an excess of 10% was lost and presumably catabolized for energy and carbohydrate anabolism. At the %CP where the highest proportion of CP was used for growth, i.e. the maximum RUE of protein for growth (slightly higher at 62%), 35% was used for body maintenance and only 3% of the excess was lost to energy.     

Greenlip abalone (Haliotis laevigata Donovan, 1808) sperm cryopreservation using a programmable freezing technique and testing the addition of amino acid and vitamin

This study investigated factors key to the development of sperm cryopreservation in the greenlip abalone Haliotis laevigata using a programmable freezing technique, including (1) permeable cryoprotectant agent (CPA) selection; (2) cooling rate; (3) endpoint temperature; (4) thawing temperature; (5) sperm to egg ratio and (6) sugar, vitamin and amino acid supplementation, using sperm motility, fertilization rate, plasma membrane integrity, mitochondrial membrane potential or acrosome integrity as quality assessment indicators. Results showed that among the permeable CPAs evaluated, 10% dimethyl sulfoxide was the most suitable for greenlip abalone sperm cryopreservation. The highest post‐thaw sperm motility was achieved with the sperm being frozen at a cooling rate of ?5°C min^?1 to ?30°C from 0°C and thawed and recovered in 40°C and 18°C seawater baths respectively. The addition of sugars in 10% dimethyl sulfoxide did not significantly improve the post‐thaw sperm motility and fertilization rate. The addition of 0.6% glycine, 0.2% taurine or 0.02% L‐ascorbic acid, on the other hand, significantly improved the post‐thaw sperm motility. However, only the addition of 0.6% glycine improved the post‐thaw sperm fertilization rate, which was further confirmed by the improvement of the post‐thaw sperm mitochondrial membrane potential and acrosome integrity through flow cytometry analysis.     

Evaluation of cytochalasin B (CB) treatments for triploidy induction in the blacklip abalone, Haliotis rubra (Leach, 1814)

The effectiveness of cytochalasin B (CB) treatments for inducing triploidy was evaluated in the blacklip abalone Haliotis rubra (Leach, 1814) in two orthogonal design experiments. The first experiment employed three dosages (DSs) of 0.25, 0.5 and 1.0 mg CB L^?1, three starting times (STs) of 5, 15 and25 min post fertilization and three treatment durations (TDs) of 10, 20 and 40 min, for a total of 27 treatments. The second experiment comprised of two DSs of 0.25 and 0.5 mg CB L^?1, five STs of 5, 15, 20, 25and 30 min post fertilization, and three TDs of 10, 20 and 40 min, for a total of 30 treatments. Water temperature was held at 17.5–18.5°C. Day 3 larvae were sampled for triploidy using flow cytometry (FCM) and survival. Optimal inductions were treatments starting at 15 or 20 min post fertilization and continuing for 40 min, and those initiated 25 or 30 min post fertilization for 20 or 40 min, using 0.5 mg CB L^?1. These treatments were all targeted at inhibition of the second polar body (PB2) formation and yielded triploidy rates of 84.8–89.5% coupled with (relative) survival rates of 20.1–52.1% in the first experiment, and corresponding rates of 86.5–96.5% and 33.0–74.1%, respectively, in the second experiment. A common and essential feature of these optimal conditions is that treatment must fully span the period of time for most of the eggs to extrude PB2. Treatments that resulted in suppression of the first polar body (PB1) formation induced triploidy levels below 71.5% and 57.6% in experiments 1 and 2 respectively. Treatments that had overlapping effects on both PB1 and PB2 extrusion led to triploidy rates above 80% but very low survival rates of 1.8% and 5.4% in experiments 1 and 2 respectively.     

Growth and feeding in juvenile triploid and diploid blacklip abalone, Haliotis rubra (Leach, 1814), at two temperatures

Growth and feeding of juvenile triploid and diploid blacklip abalone Haliotis rubra (Leach, 1814) were investigated at two temperatures of 17 and 21 °C over a 50‐day period. There were no differences in growth between triploid and diploid abalone as measured by shell length and body weight. Both triploid and diploid abalone increased in length but not in weight at 21 °C. Condition indices were similar for triploid abalone maintained at both temperatures; however, those for diploid abalone were significantly higher at 17 °C than at 21 °C. Food intake was significantly greater yet feed conversion efficiency was significantly lower in triploid than in diploid abalone. Both the feeding variables were independent of temperature. On average, diploid abalone were able to convert 1 g of dry food ingested to 0.58 g of body weight, but triploid abalone only 0.44 g.     

The effects of density on the behaviour and growth of juvenile blacklip abalone (Haliotis rubra)

The growth of juvenile abalone in aquaculture is known to be affected by density as a result of competition for food and decreases in water quality. Our results suggest that behaviour is also affected by density and this also has a significant impact on the growth of individual abalone. 1800 juveniles of Haliotis rubra were individually tagged and reared for 5 months in 12 gravity-fed tanks at two levels of density. The experimental design allowed the differentiation of the direct from the indirect effects of density. The abalone growth and distribution was monitored monthly along with the water quality. The distribution of abalone during daytime was closely related to the availability of preferred shelter space. The percentage of abalone stacked on the top of others increased with density. Preferred shelter space was characterized by low light intensity and a corner or edge for the abalone to rest against. Hides at the ends of the tanks were not occupied as much as others. Abalone were found crawling on the side of the tank during daytime when tanks were shaded. Abalone juveniles of 15–60 mm showed fidelity to their resting shelter during daytime but this fidelity was significantly reduced at the higher density. Shading of the tanks totally changed the distribution of the abalone and their daytime behaviour. Competition for shelter space reduced growth more than water quality. This revised version was published online in August 2006 with corrections to the Cover Date.     

Pharmacological and histological assessment of gut muscle movement in blacklip abalone, Haliotis rubra (Leach)

We examined the basic pharmacology of abalone gut tissues for adrenergic and peptide receptors using ligand binding and by determining the pharmacological effectors of abalone gut motility in a number of gut regions. Contractile responses could not be elicited, even though we could show the existence of α‐adrenergic and peptide receptors. Responses to any muscle contractile agents, including carbachol and potassium chloride, could not be found in blacklip abalone, Haliotis rubra (Leach). Histology confirmed the relative absence of muscular layers in all the tissues of the gut, indicating that previous literature reports of muscular involvement in contractile movement of gut contents did not appear to apply to this species.     

Effects of air exposure on the lysosomal membrane stability of haemocytes in blacklip abalone, Haliotis rubra (Leach)

The neutral red retention (NRR) assay was used to evaluate the effects of air exposure on lysosomal membrane integrity in the haemolymph of blacklip abalone, Haliotis rubra, and its subsequent recovery in water. After acclimation in 16°C water for 7 days, abalone were exposed to an air temperature of 7, 16 or 23°C for 12 h in the air exposure experiment or to these three air temperatures, e.g., for 12, 24 or 36 h, followed by re‐immersion in 16°C water in the lysosomal membrane stability recovery experiment. Statistical analyses of the air exposure experiment showed that when abalone were exposed to different air temperatures (7, 16 or 23°C), the lysosomal membrane stability was significantly affected by the air temperature, the exposure duration and their interaction. Air temperature similar to the acclimation temperature had a significantly lower impact on the lysosomal membrane stability within the initial 4.5 h in comparison with the other two temperatures in the same period. The lysosomal membrane stability recovery experiment showed that after air exposure durations of 12, 24 or 36 h, the re‐stabilization of the lysosomal membrane was faster in the animals exposed to lower temperatures than those exposed to higher temperatures. The recovery of the lysosomal membrane stability in abalone exposed to lower 7°C air temperature was not significantly affected (F_{2, 66}=0.251, P=0.779) by the exposure durations (12, 24 and 36 h) used in this study. Alternatively, the lysosomal membrane stability in abalone exposed to higher air temperatures of 16 or 23°C recovered at a faster rate when subjected to shorter durations of air exposure (F_{2, 66}=3.663, P=0.031 and F_{1, 44}=17.057, P<0.001 for 16 and 23°C respectively).     

Survival, growth and osmolality of greenlip abalone Haliotis laevigata (Donovan 1808) when exposed to different ionic profiles of inland saline water

Two experiments over 21 days were conducted to assess the effect of different ionic profiles (mainly K⁺) of inland saline water (ISW) on the survival, growth, osmoregulation and moisture content of juvenile greenlip abalone Haliotis laevigata. In experiment I, the juveniles were cultured in five different water types: ocean water (OW) as a control, inland saline water (ISW or ISW K⁺₀), ISW fortified with 33%, 66% and 100% K⁺ as in OW (ISW K⁺₃₃, ISW K⁺₆₆ and ISW K⁺₁₀₀ respectively). In experiment II, five different culture media, 100% OW(control), 75% OW mixed with 25% ISW (ISW₂₅), 50% OW mixed with 50% ISW (ISW₅₀), 25% OW mixed with 75% ISW (ISW₇₅) and 100% ISW (ISW or ISW₁₀₀), were tested. Within 21 days, all of the abalone in ISW (ISW K⁺₀ and ISW₁₀₀; Na⁺/K⁺ ratio of 111 L^?1) died compared with 100% survival in OW. One‐hundred per cent K⁺ fortification of ISW increased the specific growth rate (SGR) and serum osmolality of the surviving juveniles. The 96 h acute LC₅₀ value was between ISW₅₀ and ISW₇₅. The increased proportion of ISW in OW resulted in significantly (P<0.05) lower survival, SGR and serum osmolality of juveniles. The moisture content of the juveniles was not influenced by any water type. The results suggested that abalone cannot survive in raw ISW, but survival can increase when it is fortified with K⁺ and ionic ratios of ISW are brought closer to OW by mixing it with OW. Furthermore, abalone survival is influenced by other ions in ISW besides K⁺.     

健康和患病稚黑鲍中微生物产胞外酶的分析比较

2005年初自福建漳浦鲍鱼场患病以及健康稚黑鲍中各分离到27和56株异养细菌,在此基础上研究了它们产胞外蛋白酶、淀粉酶、明胶酶、磷脂酶及溶血能力。结果表明,虽然来自健康鲍的菌株产胞外酶的数量明显大于来自病鲍的菌株,但来自病鲍的菌株具有强分泌胞外酶能力的比例明显大于来自于健康鲍的菌株。综合考虑每一种胞外酶的潜在致病作用,以及菌株分泌多种胞外酶的能力,最后确认10株菌为潜在的致病菌。