Anaesthetic properties of a range of chemical compounds were tested on mature commercial scallops (Pectenfumatus) as a means of reducing stress and ...
Aquaculture Aquaculture 131 (199.5) 231-238
ELSEVIER
Induction of anaesthesia in the commercial scallop, Pectenfumatus Reeve Michael P. Heasman”,
Wayne A. O’Connor, Allen W.J. Frazer
NSW Fisheries, Port Stephens Research Centre, Salamander Bay, NSW 2301, Australia
Accepted 10 November 1994
Abstract Anaesthetic properties of a range of chemical compounds were tested on mature commercial scallops (Pectenfumatus) as a means of reducing stress and subsequent unplanned spawning caused by routine handling and assessment of breeding condition. Of 14 compounds tested, only chloral hydrate, magnesium chloride and magnesium sulphate successfully induced anaesthesia within 1 h. Magnesium sulphate induced high post-anaesthesia mortality and was not investigated further. Doses of 4 g chloral hydrate/l (0.024 M) or 30 g magnesium chloride/l (0.31 M) were selected as most suitable on the basis of time to, and recovery from, anaesthesia. Neither anaesthetic caused mortality nor increased spawning activity and magnesium chloride actually reduced the incidence of unplanned spawning. With the use of chloral hydrate, time to anaesthesia was found to decrease significantly with increasing water temperature in the range 12-24”C, but to be independent of temperature in the case of magnesium chloride. Keywords: Pecfen fimatus;
Anaesthetics; Spawning
1. Introduction Induced shell opening (gaping) in the scallop, Pecten fumatus, assists visual assessment of breeding condition and disease status and in the induction of spawning using intragonadal or intramuscular injections of serotonin. However, up to an hour of air exposure (emersion) is required to stimulate gaping in P. fumatus (Heasman, unpubl. obs.). This imposes considerable stress that is exacerbated by a need to place a chock between the gaping valves. Prevention of shell closure in this manner often causes physical injury to the mantle tissue and, in the worst cases, results in torn adductor muscles or rupture of the hinge ligament. * Corresponding
author. Telephone
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0044.8486/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDIOO44-8486(94)00360-2
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Protracted air exposure is frequently observed to stimulate unplanned spawning in fully conditioned (ready to spawn) P. fumatus, as is the case with other scallops such as Pecten maximus (Gruffydd and Beaumont, 1970). Even the act of transferring ripe scallops between holding tanks can trigger spawning, particularly if the temperature or other physiochemical properties of seawater, such as salinity and pH, are not closely matched. Indeed, many forms of stimulation can induce ripe scallops to spawn. These include air exposure, temperature changes as little as 1 or 2°C (Minchin, 1992), salinity changes of 5-10 mg/ kg, and exposure to sperm or high densities of microalgae, or to irritant chemicals such as potassium chloride and hydrogen peroxide (Bourne et al., 1989). The consequences of unplanned spawnings in P. fumatus following routine handling are compounded by the fact that it is an hermaphroditic species that often sheds spermatozoa and ova synchronously or in rapid succession. Useful numbers of viable eggs cannot be salvaged from such spawnings. Failure of eggs from unplanned spawnings to yield significant numbers of larvae is possibly due to high bacterial loads within conditioning units, self-fertilisation or polyspermy. Both of the latter have been shown to significantly impair the viability of developing eggs and larvae of other bivalves (Beaumont and Budd, 1983). When P. fumatus broodstock were held communally in conditioning systems, unplanned release of sperm or eggs by one or more ripe scallops also triggered the mass spawning of others in poorer condition. In some cases this included abortive release of immature eggs and fragments of ovarian tissue. There is a clear need to develop a method to allow examination of gonads and handling procedures for P. fumatus that minimise or eliminate unplanned spawning. The objective of the present study was to identify anaesthesia procedures that would both facilitate gaping and minimise handling stress and hence the incidence of unplanned spawning in hatcheryconditioned P. fumatus.
2. Materials and methods
General Adult scallops (P. fumatus) of 55-75 mm shell height were collected by divers from Jervis Bay, New South Wales (35”04’S, 150”44’E), transported to the laboratory and maintained for at least 1 week prior to experimentation. In the laboratory, scallops were held at 15°C in a temperature controlled 1400-litre recirculating system and fed microalgae to satiation. Following the week’s acclimatisation, mortality of scallops held in these recirculating systems rarely exceeds lO%/week. Seawater used in experiments, which was collected from a local coastal site, ranged in salinity from 33 to 35 g/kg. Experiments involved measuring the response of these scallops to a range of potential anaesthetics. Scallops were considered suitably anaesthetised when prodding of the mantle tissue and subsequent removal from the aquaria failed to stimulate shell closure. Continuous observation to establish the onset of anaesthesia in individual scallops was abandoned after 60 min as periods greater than this were considered impractical for routine hatchery operations. Recovery from anaesthesia was defined as the time at which shell closure in response to handling resumed and was monitored constantly for the first 90 min and every 15 min
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131 (1995) 231-238
thereafter until all scallops had recovered. Daily checks for post-anaesthesia continued for a week.
mortality were
Experiment I: Screening of potential anaesthetics A total of 14 compounds (Table 1) were tested on adult P. fumatus. Dose rates for each compound were selected on the basis of published accounts of their use on other invertebrates (Runham et al., 1965; Kaplan, 1969). Groups of 4 individually tagged scallops were placed in aerated perspex aquaria containing 4 litres of seawater at 22.0 + 0.5”C. Each group was exposed to only one anaesthetic at a single concentration. As soon as individual scallops were found to be anaesthetised, they were removed from the anaesthetising media, thoroughly rinsed in 1 pm filtered seawater and transferred to an aerated recovery tank containing 90 litres of seawater. Upon recovery, they were returned to the holding systems. Time to anaesthesia, time to recovery, behavioural response and cumulative mortalities over the following week were recorded for each compound at each concentration tested. Experiment 2: Optimum doses jbr chloral hydrate and magnesium chloride On the basis of results from Experiment 1, chloral hydrate and magnesium chloride were selected for further testing. Groups of 10 individually tagged scallops were placed in aerated perspex aquaria containing 10 litres of seawater at 20.5-22.O”C. Chloral hydrate or magnesium chloride, predissolved in seawater, was added to the aquaria to bring them to concentrations of 0.5-10.0 g/l and of 20-50 g/l respectively. The pH was measured before and after the addition of these compounds to confirm that no changes had occurred and the time to anaesthesia recorded. Once anaesthetised, scallops were transferred to an aerated Table I Compounds ,fionatus
used in preliminary
Compound
screening tests for the induction of anaesthesia
Dose 0.1, I g/l
Aspirin Benzocaine Chloral hydrate Ethanol Isobutynol Magnesium chloride Magnesium sulphate
0.1. 1 g/l 2.5.5, IO g/l 1% 0.5, I ml/l IO, 20, 30 g/l 20, so, 80 g/l
Metomidate MS222
20,200 mgil 0.1, 1 g/l
Nembutal Phenoxy ethanol Quinaldine sulphate Tertiary amyl alcohol Valium
15 mg/l 0.6 ml/l 2.5.2.50 mg/l 0.1. 1 ml/l 5, 10, IS, 20, and 40 mgll
Comments No apparent effect Initial hyperactivity Anaesthesia No apparent effect No apparent effect Anaesthesia Anaesthesia followed by high mortality Shell closure Hyperactivity and hyperextension of tentacles No apparent effect No apparent effect Shell closure No apparent effect No apparent effect
in the commercial
scallop, Pecten
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recovery tank and continuously flushed with filtered seawater. All subsequent and handling procedures were as described for Experiment 1.
monitoring
Experiment 3: Effects of temperature on anaesthesia induced by magnesium chloride and chloral hydrate Four replicate aquaria were set up at each of 4 temperatures ( 12,16,20 and 24°C). Three individually tagged scallops were placed in 3 litres of seawater in each aquarium and treated with chloral hydrate (4 g/l, 0.024 M) as described for Experiment 2. All subsequent monitoring and handling procedures were also as described for Experiment 2. These procedures were repeated with different scallops using magnesium chloride (30 g/l, 0.3 1 M). Experiment 4: Effects of anaesthesia on handling induced spawning in ripe scallops Thirty P. fumatus in prime spawning condition were removed from a broodstock conditioning system and placed individually in aquaria containing 10 litres of seawater. The temperature of each aquarium was maintained at 16.5”C, which was the same as that of the conditioning system. Chloral hydrate (4 g/l) was added to 10 of the aquaria, magnesium chloride (30 g/l) to a further 10 and the remaining 10 aquaria were maintained as “untreated controls”. Time to anaesthesia was recorded and scallops were then transferred to an aerated recovery tank as previously described. Following recovery, scallops were placed in aerated aquaria containing filtered seawater at 16.5”C and monitored for 48 h to determine if spawning occurred. Five days later all scallops were again visually assessedfor reproductive condition. During routine hatchery operations, another4 batches of 20 ripe P. fumatus were subjected to visual assessment of gonad condition. Half the scallops from each batch were anaesthetised using magnesium chloride to induce gaping while the remainder were emersed until gaping occurred. The number of individuals spawning during the following 24 h was recorded. Statistical analysis Data from Experiment 3 were analysed using single-factor ANOVA to test the effect of temperature on time to anaesthesia. Homogeneity of variances was tested using Cochran’s test and means were compared using Tukey’s honestly significant difference method (Winer, 1971). A chi-squared test (Winer, 1971) was used to determine if the use of magnesium chloride significantly decreased the incidence of unplanned spawning of scallops after handling and air exposure. To determine whether scallop size affected the relationship between dose and time to anaesthesia, an analysis of covariance (with scallop size as a covariant) was conducted using pooled data for magnesium chloride (30 g/l) from Experiments 2 and 3 and pooled data for chloral hydrate (4 g/l) from Experiments 2 and 3.
3. Results Of the 14 anaesthetics tested at various concentrations in Experiment 1, only magnesium chloride, magnesium sulphate and chloral hydrate induced anaesthesia within 60 min (Table
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Table 2 The effects of chloral hydrate and magnesium
131 (1995) 231-238
chloride dose rates upon anaesthesia
235
in the commercial
scallop,
Pectenfundus Time to anaesthesia ( min ) ’
Time to recovery (min)
Mortality at 24 h
Chloral hydrate 0.5 1.I5 2.5 3 4 5 I 8.5 10
ineffective 36.5 f 21.5 56.6544.9 29.5 + 14.6 12.9f6.4 22.2 + 12.6 7.9 + 2.9 8.8 * 2.3 7.5 * 1 .o
_ 32.5 *31.3 61.4+43.5 52.8 f40.2 28.0+ 11.4 31.5 * 29.9 20.5 f 14.3 > 120 _
0 0 10 0 0 40 20 70 100
Magnesium 20 25 30 35 50
44.5 * 18.9 12.5 f 10.6 4.8f3.3 3.S_+ 1.8 3.653.6
Anaesthetic
dose (g/l)
(%)
chloride
‘Values are means fs.d.,
8.2 f4.7 4.9* 1.9 4.3 * 3.9 2.35 1.5 4.2 f 3.2
0 0 0 0 0
n = 10 for all dose rates except chloral hydrate 10 g/l where n=4.
1) . Magnesium sulphate was not tested further because of the high concentration (0.66 M, 80 g/l) required to induce anaesthesia and high post-anaesthesia mortality ( > 90%) at the effective dose. The response of scallops to various concentrations of chloral hydrate and magnesium chloride in Experiment 2 are presented in Table 2. Doses of 4 g/l chloral hydrate and 30 g/l magnesium chloride were adopted as standards for subsequent trials on the basis that these were the lowest concentrations that induced rapid anaesthesia without subsequent mortality. Scallop size, in the range 55-75 mm shell height, had no significant effect (P > 0.05) on time to anaesthesia. Recovery from chloral-hydrate-induced anaesthesia varied widely within treatments, ranging from 4 min to greater than 90 min. In contrast, all scallops anaesthetised with magnesium chloride recovered within 10 min regardless of concentration (Table 2). Significant differences were found in the time taken for chloral hydrate to induce anaesthesia in the range 12-24°C (F = 4.23, df = 15, P < 0.05)) while regression analysis of the sample means showed significant correlation between temperature and time to anaesthesia ( r2 = 85, P < 0.05), Magnesium-chloride-induced anaesthesia was not affected by temperature in the range 12-24°C (Fig. 1). No mortality occurred following exposure to either compound at the concentrations and temperatures used. In Experiment 4,20% (2 of 10) of ripe scallops anaesthetised with magnesium chloride and 30% of those anaesthetised with chloral hydrate spawned within 7 days of handling. A chi-squared analysis of spawning frequency data obtained during routine hatchery operations indicated that, compared with air exposure, the use of magnesium-chloride-induced
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‘\
“
‘, \
_ l. l,,
sl
p it
lo0
mqneslum chloride I
I
I
I
I
12
16
20
I
I
I
24
Temperature (“c) Fig. 1. The effect of temperature upon time to anaesthesia are means (n = IO) and bars are standard errors.
using magnesium
chloride or chloral hydrate. Points
anaesthesia to elicit gaping and assess breeding condition significantly the incidence of subsequent unplanned spawning from 38 to 10%.
decreased (P < 0.05)
4. Discussion Of the 14 chemical compounds tested, three were found to induce anaesthesia in adult P. fumatus within a practical time limit of 60 min. One of the three, magnesium sulphate, which has previously been shown to induce anaesthesia in a variety of molluscs, including the European flat oyster, Ostrea edulis (Culloty and Mulcahy, 1992) and freshwater and marine snails (Knudsen: in Kaplan, 1969)) was lethal to P. fumatus at effective concentrations. Toxicity of magnesium sulphate to juvenile abalone (Huliotis gigantia) was also reported by Sagara and Ninomiya ( 1970)) although much larger doses (300 g/l) and longer exposure times ( > 2 h) were involved. The remaining two effective compounds, chloral hydrate and magnesium chloride, proved much safer alternatives. During this study, a combined total of 106 individual P. fumatus were exposed to anaesthetising doses of chloral hydrate of 1.75-4 g/l. A total of 132 scallops were exposed to anaesthetising doses of 30-50 g/l magnesium chloride. Of these, only one scallop, which had been exposed to 2.5 g/l chloral hydrate, died within 7 days of anaesthesia. Moreover, no other adverse side-effects such as persistent gaping, aberrant extension or retraction of tentacles and mantle tissue, retardation of the shell closure reflex or loss of breeding condition in the absence of spawning, were observed when chloral hydrate and magnesium chloride were used at optimal anaesthetising concentrations of 4 g/l and 30-50 g/l respectively. Both magnesium chloride and chloral hydrate have been shown to depress beating of lateral gill cilia in the American oyster, Crussostrea virginica (Galtsoff, 1964). Magnesium chloride was used by Whyte and Carswell (1983) to induce gaping in the Pacific oyster Crassostrea gigas. Gaping was considered by these authors to be a product of inhibited muscular contraction due in turn to displacement of calcium from, and infusion of magne-
M.P. Heasrnan et al. /Aquaculture 131 (1995) 231-238
231
sium into, the tissues. Optimal dose rates of 0.327 M (32 g/l) magnesium chloride for C. gigas (Whyte and Carswell, 1983) and 35 g/l magnesium chloride for 0. edzdis (Culloty and Mulcahy, 1992) were very similar to those found for P. jiimatus (30 g/l) in the present study. The failure of anaesthetised P. fumatus to respond to tactile stimulation may have resulted from the inhibition of synaptic mechanisms by magnesium chloride. Stephens (1978) found that pallial nerve activity and reflex movements of the mantle in scallops were reversibly blocked by soaking in calcium-free, high-magnesium solutions. Magnesium chloride at 30 g/l has now been adopted for routine use on scallops in our laboratory on the basis that it provides much more rapid and consistent induction of and recovery from anaesthesia (Table 2) than chloral hydrate. Moreover, scallops appear far more tolerant to overdoses of magnesium chloride, recovering rapidly after exposure to almost twice the optimal dose rate and after being exposed to the optimal dose rate for periods exceeding 1 h. The latter is more than 10 times the mean duration required to induce anaesthesia. A further advantage of magnesium chloride over chloral hydrate for the induction of anaesthesia is that the former is not significantly affected by the temperature in the range 12-24°C (Fig. 1) . This temperature range approximates both seasonal oceanic temperatures along the southern coast of NSW and ambient temperatures encountered within our laboratory. While neither magnesium chloride nor chloral hydrate totally prevent spawning associated with handling of ripe scallops, their use facilitates inspection of the gonad and other soft tissues without the risk of damage to the mantle, adductor muscle or hinge ligament. Anaesthesia of scallops also eliminates the risk of desiccation associated with air exposure when used to induce gaping.
Acknowledgements The authors thank Wayne Walker and John Kelly for the collection of scallops as well as the staff of the Port Stephens Research Centre for their assistance in the preparation of this manuscript. In particular we applaud Dr. Geoff Allan, Dr. John Nell, Mr. Bill Talbot and the anonymous referees for valuable editorial comments. This study was undertaken as part of a Fishing Industry Research and Development Trust Fund grant 91/53.
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Minchin, D., 1992. Induced spawning of the scallop, Pecten maximus, in the sea. Aquaculture, 101: 187-190. Runham, N.W., Isarankura, K. and Smith, B.J., 1965. Methods for narcotizing and anaesthetizing gastropods. Malacologia, 2 ( 2) : 23 I-238. Sagara, J. and Ninomiya, N., 1970. On the tear off of young abalone from the attachment by four anesthetics (ethyl carbamate, magnesium sulfate, chloral hydrate and sodium diethylbarbiturate). Suisan Zoshoku, 17(2): 89. Stephens, P.J., 1978. The sensitivity and control of the scallop mantle edge. J. Exp. Biol., 75: 203-221. Whyte, J.N.C. and Carswell, B.L., 1983. Chemical aid for shucking the Pacific oyster, Crassostrea gigas. Can. Tech. Rep. Fish. Aq. Sci., 1238: 38 pp. Winer, B.J., 1971. Statistical Principles in Experimental Design. McGraw-Hill Kogakusha, Tokyo, pp. 205-210.
