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.     

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.     

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.     

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.     

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.     

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.     

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.     

Assessment of the physiological effect of altered salinity on greenlip (Haliotis laevigata) and blacklip (Haliotis rubra) abalone using respirometry

The early physiological response (3 days) of greenlip (Haliotis laevigata Leach) and blacklip (H. rubra Leach) abalone to a single abrupt change in salinity was monitored using a multi‐channel open‐circuit respirometer. The range of salinity tolerance for both species in this trial was 25–40 ppt. Indications from other trials were that a margin of 2 ppt outside this range will cause mortality. Serum volume was increased by reduced salinity as much as 25% in the short term, with equilibration of the concomitant whole body weight increase (9.2±0.5%) occurring within 1 h. The serum volume appeared to be decreased at high salinity. There was little underlying change in basal oxygen usage levels, but significant behavioural changes that affected overall oxygen consumption. Both high and low salinity appeared to reduce activity. Animals in low salinity exhibit partial recovery of activity levels after 1 day in a manner similar to other stress responses. Animals at high salinity (40 ppt) did not show recovery of activity levels over 3 days. Overall, these results suggest that greenlip and blacklip abalone will have little trouble tolerating moderately low‐salinity environments.     

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.     

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.     

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).     

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.     

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.     

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.     

The effects of protein kinase A catalytic subunit on sperm motility regulation in Pacific abalone Haliotis discus hannai

Protein kinase A plays a central role in the regulation of sperm motility from echinoderms to mammals, but the information about its regulatory role in molluscs is very limited. In this study, a protein kinase A catalytic subunit (designated as HdPKA‐C) was identified from Pacific abalone Haliotis discus hannai. The open reading frame of HdPKA‐C was of 1,077 bp, encoding a peptide of 358 amino acids with a typical protein kinase domain. HdPKA‐C shared 82%–87% sequence similarities with other PKA‐Cs, and it was clustered first with gastropod PKA‐Cs in the phylogenetic tree. The mRNA of HdPKA‐C was constitutively expressed in examined tissues, with the highest level detected in hepatopancreas. The phosphorylated form of HdPKA‐C (p‐HdPKA‐C) was localized at the acrosome, connecting piece and flagellum of spermatozoa with variable intensity. Its phosphorylated substrates were also detected in these regions with much lower intensity at the connecting piece. The inhibition of HdPKA‐C activity with H‐89 led to a significant reduction in the percentage of motile sperm and sperm velocities. p‐HdPKA‐C was detected by Western blot in strip‐spawned sperm, naturally spawned sperm and H‐89‐treated sperm with almost the same intensity. The intensity of p‐HdPKA‐C substrates in naturally spawned sperm was higher than that in strip‐spawned sperm, and it was roughly the same as that in H‐89‐treated sperm except for two bands at 50 and 60 kDa. These results collectively indicated that HdPKA‐C played an important role in the regulation of abalone sperm motility by altering its substrates phosphorylation.     

Evaluation of an in vitro digestibility technique for the prediction of protein digestibility in the South African abalone, Haliotis midae L.

A pH-stat multienzyme in vitro digestibility assay was investigated for its efficacy in predicting apparent protein digestibility coefficients in abalone diets. Linear regression analysis between in vitro digestibility estimates and in vivo digestibility coefficients obtained from abalone, revealed that the technique could be used to reliably predict apparent protein digestibility. Maximal predictability of the system was obtained when protein sources were analysed according to origin – animal or plant. The technique was used to assess the apparent protein digestibility of 34 potential protein sources for use in formulated feeds for Haliotis midae .     

Reduced water oxygen levels affect maximal feed intake, but not protein or energy utilization efficiency of rainbow trout (Oncorhynchus mykiss)

This study examined the effect of reduced water oxygen levels on the utilization efficiencies of energy and protein from a diet fed to rainbow trout. An experimental diet was fed at one of the four ration levels with an additional starved treatment also included in each oxygenation regime. Oxygen levels in each oxygenation regime varied with ration level, but averaged 9.3 ± 0.36 mg L⁻¹ for the normal regime and 5.7 ± 1.4 mg L⁻¹ for the hypoxia regime. Significant differences were observed in the apparent satietal feed intake levels in each oxygenation regime, but not at any of the pair-fed restricted levels. No significant effects of oxygenation regime were observed on the utilization of either energy or protein by the fish. Efficiency of protein use varied depending on the protein intake level, but was not significantly affected by oxygenation regime. This study demonstrates that a reduction in the oxygen levels of the water does not affect the utilization efficiency of dietary digestible protein and energy in rainbow trout, but does result in a downregulation of feed intake when the fish is fed to apparent satietal levels.     

Effect of dietary protein and temperature on the growth and health of juvenile New Zealand black‐footed abalone (Haliotis iris)

The protein requirements for optimal growth and health of juvenile New Zealand black‐footed abalone (Haliotis iris) under different water temperatures were investigated. Six diets with different crude protein amounts (0, 10, 20, 30, 40 and 45%) and two temperature regimes (13–21 and 8–16 °C) were used to culture juvenile abalone over a 4‐month period. Growth (shell lengths and animal weights) and health (survival, activity and mucous production) parameters were recorded for animals within all protein and temperature combinations. Proximate analyses and amino acid profiles were also performed on the diets, soft bodies (including gonad), and shells to evaluate the overall nutrient contents (diet and animal) and requirements (animal). The results indicate that the protein requirements for juvenile H. iris are higher when the environmental temperatures are low. Thus, increasing the dietary protein level results in better growth [i.e. increase in soft body (including gonad) crude protein and mean protein gain] and health (i.e. more goblet cells and thicker epithelial layers). Results from amino acid profiles in abalone soft bodies (including gonad) and shells show that these parameters did not change considerably with different dietary protein levels, but temperature did affect the concentration of several amino acids in both soft bodies (including gonad) and shells.     

Comparative performance of juvenile red abalone, Haliotis rufescens, reared in laboratory with fresh kelp and balanced diets

Juvenile Haliotis rufescens were reared in the laboratory in order to investigate the extent to which fresh kelp and formulated feeds with 250 g kg⁻¹ (25P) and 380 g kg⁻¹ protein content (38P) affected their growth rate, gut residence time (GRT), food consumption ( C ), food conversion ratio (FCR) and digestibility. Abalone from 38P attained the highest growth rate (70.5 ± 4.2 μm day⁻¹; 98.3 ± 6.95 μg day⁻¹), followed by 25P (47.9 ± 2.79 μm day⁻¹; 67.4 ± 2.82 μg day⁻¹) and kelp (23.6 ± 3.36 μm day⁻¹; 28.2 ± 4.11 μg day⁻¹). No significant differences were observed in consumption rate among treatments (0.61–0.68% body weight per day), yet kelp-fed abalone exhibited higher FCR (2.44), protein efficiency ratio (4.42), and apparent digestibility of dry matter (69.5%), protein (69.8%) and gross energy (79.2%) than 38P organisms (59.8, 62.4 and 62.2%, respectively). They also showed longer GRT (23.1 ± 0.93 h). This study demonstrated that formulated diets with 250 g kg⁻¹ and 380 g kg⁻¹ protein inclusion can sustain higher growth rates of juvenile H. rufescens than fresh algae. These differences seem to be due to the amount of dietary protein. Kelp meal appears to improve the consumption and digestibility of balanced diets, and its inclusion in formulated diets is recommended.     

Partial utilization efficiencies of protein and methionine by barramundi (Lates calcarifer) in response to dietary methionine source and form

An experiment was conducted with barramundi (Lates calcarifer) juveniles (initial weight 10.3 g ± 0.3 g) to examine the partial efficiency of utilization of methionine (Met) from intact protein (fishmeal or lupin protein concentrate) and a crystalline DL‐Met source. Fish were fed at one of three ration levels: Low (0.4 g/fish/day), Moderate (0.8 g/fish/day) and High (1.6 g/fish/day). Those fed the fishmeal‐based diet (Diet FML) at the highest ration level grew to an average weight of 37.3 ± 0.46 g, whereas those fed the Lupin Protein Concentrate (LPC)‐based diet fortified with all EAA (Diet LPCM) at the highest ration level grew to 25.4 ± 2.27 g. The weight of the fish fed the LPC diet with no additional Met (Diet LPC) even at the highest ration level declined over the course of the experiment resulting in a final weight of 9.2 ± 0.88 g, clearly demonstrating the impact of dietary Met deficiency. The partial efficiency of protein utilization was also significantly reduced when Met was limiting (a coefficient of 0.06, compared to 0.39 in the Met‐supplemented LPC diet). The results suggested that the partial efficiencies (coefficients) of both Met and protein utilization in diets where crystalline Met is the primary source of Met (Diet LPCM: 0.26 and 0.39 respectively) were significantly poorer than from an intact protein source (Diet FML: 0.89 and 0.67 respectively) when Met is provided in excess.