Use of anaesthetics with Pinctada maxima
To produce a cultured round pearl, a skilled-technician must implant a nucleus into the gonad of a recipient pearl oyster together with a piece of mantle tissue from a sacrificed donor oyster (Gervis & Sims, 1992). Subsequent proliferation of the donor mantle tissue forms the pearl-sac around the nucleus, and deposition of nacre from the pearl-sac onto the nucleus forms a cultured pearl over a period of about 2 years (Acosta-Salmon et al., 2004; Gervis & Sims, 1992).
To optimise pearl quality, pearl oysters must be treated appropriately to minimise stress during and after the pearl implantation procedure which may include forced opening of their shells and incision of the gonad prior to implantation. Anaesthetics have been investigated as a means of reducing stress and mortality of pearl oysters resulting from pearl implantation (Norton et al., 2000). In a more recent development, anaesthetics were used to enable removal of mantle tissue from donor pearl oysters without killing them (Acosta-Salmon & Southgate, 2005; Acosta-Salmon & Southgate, 2006; Acosta-Salmon et al., 2004).
This potentially allows oyster donors that produce high quality pearls to be used as future broodstock. Furthermore, pearl oysters readily regenerate excised mantle tissue (Acosta-Salmon and Southgate, 2005; Acosta-Salmon and Southgate, 2006) and so donor oysters that are anaesthetised for mantle tissue removal, rather than killed, could potentially be used for pearl implantation on more than one occasion (Acosta-Salmon et al., 2004). This approach offers considerable benefits to the cultured pearl industry and justifies further investigation of the use of anaesthetics with pearl oysters.
The response of pearl oysters to anaesthetics has been shown to vary between species according to the type and concentration of the anaesthetic used. Propylene phenoxetol at a concentration of 2-3 mL L-1 has been used successfully with Pinctada albina, P. imbricata, P. margaritifera and P. maxima (Norton et al., 1996; O’Connor & Lawler, 2002). Benzocaine at a concentration of 1200 mg L-1 was similarly used to successfully induce relaxation in P. albina, P. margaritifera and P. fucata over a short period of time and with a short recovery time (Acosta-Salmon et al., 2005; Norton et al., 1996); however, this anaesthetic was less effective at a lower concentration of 500 mg L-1 (Acosta-Salmon et al., 2005). More natural derivatives, such as clove oil and menthol, have also shown a degree of effectiveness in inducing relaxation in pearl oysters (Norton et al., 1996). Prolonged exposure to an anaesthetic may cause the mantle or body of pearl oysters to lose rigidity and collapse (Acosta-Salmon et al., 2005; Mills et al., 1997; Norton et al., 1996; O’Connor & Lawler, 2002). It may also result in mantle retraction and excessive mucus production (Norton et al., 1996) and render pearl oysters unsuitable for pearl implantation. Anaesthetics are difficult to manage under large-scale pearl implantation conditions and they have not yet been widely applied in the cultured pearl industry (Acosta-Salmon et al., 2005). However, donor pearl oysters are required in much smaller numbers than recipient oysters during the implantation procedure, and the use of anaesthetics with donors alone would be a more manageable proposition. Given the potential benefits of using anaesthetics with donor pearl oysters, there is a need for further research in this field with a view to overcoming some of the potential problems outlined above. With this objective, this study determined the effectiveness of five anaesthetics, at varying concentrations, in inducing relaxation in P. maxima.
2.2 Material and methods
The oysters used in this study had a mean (± SD) dorso-ventral measurement (DVM) of 128.9 ± 12.5 mm and were maintained in suspended culture at James Cook University’s Orpheus Island Research Station off Townsville, north Queensland, Australia. They were cleaned and maintained in a raceway prior to the experiment. Twenty-seven oysters were randomly distributed between three replicate 20 L aquaria used for each of seven anaesthetic treatments: 3 mL L-1 of 2-phenoxyethanol (Sigma-Aldrich, Inc.), 1.5 mL L-1clove oil (Continental Flavour), 0.25 mL L-1 menthol liquid (Auroma Pty Ltd.), 0.4 mL L-1 menthol liquid, 2.5 mL L-1 of propylene phenoxetol (Nipa Laboratories Ltd.), 500 mg L-1 and 1200 mg L-1 of benzocaine (Sigma-Aldrich, Inc.). Thus each aquarium contained nine oysters. A further set of aquaria was used as controls and contained oysters held in 1 µm filtered sea water. The concentrations of the particular anaesthetics used in this experiment were based on those used by Norton et al. (1996).
To prepare the solutions of propylene phenoxetol, 2-phenoxyethanol, clove oil and menthol liquid, each was added to seawater in a small container and shaken vigorously before being transferred to a large container of seawater (Norton et al., 1996). The 500 mg L-1 solution of benzocaine was made using a preparation of 1:4 w/v benzocaine:methanol solution that was poured into hot seawater, before being transferred into a large container of seawater to reach the desired concentration (Acosta-Salmon et al., 2005). To prepare the 1200 mg L-1 benzocaine solution, benzocaine was dissolved in ethanol (100 g L-1) and the resulting solution was mixed slowly into seawater (Acosta-Salmon et al., 2005).
Aquaria were filled with 1 µm filtered sea water and the respective anaesthetic solutions were added to give the desired final concentrations. The temperature of seawater in the aquaria was maintained at 24 ± 1ºC and pH ranged from 8.0 to 8.2. Prior to placing the oysters into the aquaria, the pH of each aquarium was recorded. Oysters used in the experiment were first placed hinge-down in a tray until their shells opened. A plastic wedge was placed between the shell valves to prevent closure and allow rapid access of anaesthetic solution to oyster tissues once they were placed into the aquaria. Oysters were placed into mesh baskets which were then suspended into the aquaria.
Once exposed to the anaesthetic solutions, oysters were observed continually. Oysters were considered to be relaxed when they no longer responded to stimulation (touching) of the mantle tissue (Norton et al., 1996). The timing of relaxation and the proportion of relaxed oyster in each aquarium were recorded for a 30 minute period which began when the first oyster in each aquarium became relaxed. Within this period oysters were also observed for ‘body collapse’ and ‘mantle collapse’ (Acosta-Salmon et al., 2005). Mantle collapse is characterised by mantle tissue that falls away from the shells, and body collapse is characterised by lack of muscular strength in all soft body parts (Acosta-Salmon et al., 2005).
In both cases, they are considered unsuitable as donor oysters for pearl implantation (Acosta- Salmon et al., 2005). Relaxed oysters that did not show these characteristics were categorised as suitable donor oysters. The number of suitable donors within each anaesthetic treatment was recorded throughout the 30 min observation period. Oysters with mantle or body collapse were removed from aquaria to a raceway containing running seawater. All other oysters were retained in treatment aquaria for the 30 min period and then transferred to running seawater where their recovery was monitored for a further 2 h. Oysters were considered to have recovered when they closed their shells in response to touching of their mantle tissue (Norton et al., 1996). They were then placed into panel nets (Gervis & Sims, 1992) and transferred to a long-line culture system in the ocean. Oyster survival was recorded for a further month after exposure to anaesthetics.
A Kruskall-Wallis (κ) analysis was used to determine whether there was a difference between anaesthetics in terms of the time required for oysters to relax. A Pearson’s Chi Square (χ2) test was conducted to assess the effectiveness of anaesthetics based on the number of relaxed oysters and oyster survival. The Kruskall-Wallis analysis was generated by SPSS ver. 13, but the Pearson’s Chi Square was by Analyse-it ver. 2.11 for Microsoft Excel 2003.
The mean times required for oysters to become relaxed and to recover from exposure to anaesthetics are shown in Table 2.1. Oysters exposed to 1200 mg L-1 benzocaine showed the fastest time to relaxation of 10.5 (± 7.9) min while those treated with 0.4 mL L-1 menthol liquid required the longest time of 31.3 (± 5.2) min to reach relaxation. Oysters exposed to 3 mL L-1 2-phenoxyethanol reached relaxation at 13.8 (± 6.4) min and this anaesthetic resulted in the highest proportion of relaxed oyster (96.3%) of all anaesthetics tested. Although both 2.5 mL L-1 propylene phenoxetol and 1200 mg L-1 benzocaine brought about relaxation of 88.9% of oysters, they showed differences in induction time with 2.5 mL L-1 propylene phenoxetol achieving this in 15 (± 7.1) min compared to 10.5 (± 7.9) min for 1200 mg L-1 benzocaine. The lowest proportion of relaxed oysters (51.9%) was recorded in the 500 mg L-1 benzocaine treatment even though the average time to relax was relatively low (17.5 ± 8.9 min). Oysters exposed to 0.25 mL L-1 menthol liquid did not relax. All but four of the oysters exposed to clove oil became relaxed, but all relaxed oysters in this treatment died during the 30 min observation period. Death was preceded by production of excessive mucus and mantle retraction. This condition was followed by mantle and body collapse.
Within the 30 min observation period following relaxation of the first oyster in each aquarium, all treatments experienced a decrease in the number of suitable donor oysters with the exception of the 500 mg L-1 benzocaine treatment (Fig. 2.1). The number of suitable donor oysters decreased after 20 min in both the 2-phenoxyethanol and 0.4 mL L-1 menthol liquid treatments, after 25 min in the 1200 mg L-1 benzocaine treatment, and after 15 min when exposed to propylene phenoxetol. These decreases resulted from the onset of mantle or body collapse in relaxed oysters. However, numbers of suitable donors following exposure to 2-phenoxyethanol, 1200 mg L-1 benzocaine and propylene phenoxetol increased rapidly to more than half the total in each treatment (total 27 oysters) within 10 min (Fig. 2.1). The highest numbers recorded were 19 suitable donors (70.3%) for both 2-phenoxyethanol and propylene phenoxetol treatments, and 21 suitable donors (77.8%) in the 1200 mg L-1 benzocaine treatment (Fig. 2.1).
All oysters in all treatments, except those exposed to clove oil, reached normal condition within two hours of removal from exposure to the various anaesthetics. One month later, nearly 100% survival of oyster was observed in the treatments using 3 mL L-1 2-phenoxyethanol, 500 mg L-1 benzocaine, 2.5 mL L-1 propylene phenoxetol, and the control, with only one dead oyster in each of them (Fig. 2.2). Six dead and three dead oysters resulted from exposure to 0.4 mL L-1 menthol liquid and 1200 mg L-1 benzocaine, respectively. Although all oysters that relaxed after being exposed to clove oil died shortly after exposure, the four oysters in this treatment that did not relax were still alive after one month (Fig. 2.2). With the exception of the clove oil treatment, survival of oysters from the remaining
anaesthetics did not differ significantly
The results of this study show that benzocaine at a concentration of 1200 mg L-1, 2-phenoxyethanol and propylene phenoxetol were among the better anaesthetics to use for P.maxima. A summary of the effectiveness and characteristics of these three anaesthetics is shown in Table 2.2. Each varied in the time required to induce relaxation, the proportion of oysters that relaxed, the time that oysters remained relaxed, the time required for relaxed oysters to recover, and oyster survival. Norton et al. (1996) suggested that anaesthetics used with pearl oysters should ideally induce relaxation in less than 15 min and allow rapid recovery of oysters following anaesthesia (< 30 min). While, benzocaine (1200 mg L-1), 2-phenoxyethanol and propylene phenoxetol did induce relaxation within 15 min, none of them allowed recovery within 30 min. Different species of mollusc react differently to particular anaesthetics and concentrations (Aquilina & Roberts, 2000; Araujo et al., 1995) and a summary of a number of studies in this field is shown in Table 2.3.
The induction time for P. maxima exposed to 1200 mg L-1 benzocaine in this study was similar to those observed for both P. margaritifera and P. fucata in a prior study (Acosta-Salmon et al., 2005). Furthermore, relaxation of P. maxima following exposure to 2.5 mL L-1 propylene phenoxetol was also similar to that observed for P. margaritifera when exposed to similar concentration of the same chemical (Norton et al., 1996). In contrast, a concentration of 2.5 mL L-1 propylene phenoxetol caused high mortality of the abalone, Haliotis iris (Aquilina & Roberts, 2000)(Table 2.3). P. maxima showed much slower relaxation (13.8 min) when exposed to 2-phenoxyethanol than reported for the abalone, H. midae (White et al., 1996). Furthermore, neither 2-phenoxyethanol or benzocaine, which were effective anaesthetics for P. maxima in this study, were successful in inducing relaxation in a recent study with the queen conch, Strombus gigas (Acosta-Salmon & Davis, 2007)
In this study, the three anaesthetics that induced the highest proportions of relaxed oysters (1200 mg L-1 benzocaine, 2-phenoxyethanol and propylene phenoxetol) also resulted in oysters which remained in a relaxed state for longer periods (> 15 mins) before showing signs of mantle and body collapse, when compared to the other chemicals tested. This is an important factor when considering the potential use of relaxed oysters in the pearl implantation process, because it increases the period over which an oyster can be utilised as a mantle tissue donor. An inability to maintain this condition, i.e. oysters showing mantle or body collapse or even mortality, renders an oyster unsuitable as a donor (Acosta-Salmon et al., 2005).
Anaesthetics may disrupt synaptic transmission in the neural system of molluscs (Spencer et al., 1995; Woodall et al., 2003) and longer periods of exposure to anaesthetics may lead to neuro-degeneration of the important organs within animals (Woodall et al., 2003) and subsequently death. The death of a large proportion of the P. maxima exposed to clove oil indicates a toxic nature to this chemical. Besides changes in the morphological appearance of their soft tissues, affected oysters also produced excessive mucus. Norton et al. (1996) recorded mucus production from P. albina following exposure to the anaesthetic MS222, but not when exposed to clove oil at the same concentration as used in the present study.
Clearly there are differences in the degree of toxicity of clove oil to various pearl oyster species. However, it is interesting to note that four of the 27 P. maxima exposed to clove oil in this study did not relax or have an adverse reaction to this chemical; they were still alive one month after exposure to clove oil. Mantle tissue of the P. maxima that died following exposure to clove oil became inflamed and more red in colour before developing lesions indicating the death of mantle epithelial cells. This epithelial irritation may have resulted from too high a concentration of clove oil and it is possible that this chemical may be effective as an anaesthetic for P. maxima at a lower concentration. The death of some oysters recorded a month after exposure to menthol liquid also raises concerns about the toxicity of this chemical to P. maxima and its use as an anaesthetic. On the basis of our results, we do not recommend the use of clove oil or menthol liquid as anaesthetics for P. maxima.
Other important considerations when assessing the effectiveness of anaesthetics include their ease of use and preparation and potential toxicity to human users. All chemicals chosen in this study have low risk to human health. However, particular anaesthetics require more effort in preparation than others and may vary in their solubility in seawater. These characteristics are outlined for 2-phenoxyethanol, 1200 mg L-1 benzocaine and propylene phenoxetol in Table 2.2. These three chemicals were effective anaesthetics for P. maxima as outlined above, however, they varied in their preparation and solubility (Table 2.2). While 2- phenoxyethanol, benzocaine and propylene phenoxetol may be considered the most suitable of the anaesthetics tested for P. maxima, 2-phenoxyethanol and propylene phenoxetol are perhaps superior because of their ready solubility in seawater and resulting ease of preparation. While this experiment has identified anaesthetic treatments that produce relaxation in P. maxima and will facilitate mantle excision, this procedure is only useful if there are no adverse effects on pearl production, when mantle tissue from anaesthetised donor oysters is used for pearl implantation. Subsequent research will address this issue (Chapter 5 and 6).
Article source: Mamangkey, Noldy (2009) Improving the quality of pearls from Pinctada maxima. PhD thesis, James Cook University.
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