Elsevier

Aquaculture

Volumes 422–423, 20 February 2014, Pages 202-210
Aquaculture

Histopathology and haemolymph biochemistry following anaesthesia and movement in farmed Australian abalone (Haliotis rubra × Haliotis laevigata)

https://doi.org/10.1016/j.aquaculture.2013.11.035Get rights and content

Highlights

  • We identify biochemical and histopathological changes due to benzocaine anaesthesia.

  • Stress effects were seen in haemolymph and the left kidney and the foot was ulcerated.

  • Quantitative methods allowed differentiation of damage in tissues from background lesions.

  • Comparison of haemolymph changes with histopathology, to show if non-destructive sampling can indicate tissue damage.

  • The methods can be applied to all on-farm procedures in order to improve husbandry.

Abstract

Haemolymph and tissue samples of Haliotis rubra × laevigata hybrid abalone were taken during a routine stock movement procedure on an Australian abalone farm to look for biochemical and histological changes associated with anaesthesia and/or manual movement of the abalone, both of which are used in commercial stock movement operations. Sections of the left kidney, the gill and the surface of the foot were examined under light microscopy and a scoring system was used to measure observed changes and compare treatment groups to controls. The left kidney of anaesthetised abalone contained less protein and haemocytes than control or manually moved abalone on the day of anaesthesia (p < 0.05), but returned to baseline levels within 1 day. The foot muscle of anaesthetised and moved abalone had areas of denuded epithelium 5 days after anaesthesia (p < 0.05). Protein, phosphate, magnesium, sodium, chloride, calcium and potassium levels were measured in the cell-free haemolymph. The protein levels were significantly elevated compared to controls on the day after anaesthesia and manual movement (p < 0.05), but declined to baseline levels within 3 days. There were no significant differences in the other measured ions. Benzocaine anaesthesia, with or without subsequent movement, led to greater histological changes in tissues of abalone, than manual movement without anaesthetic. Comparison of physiological and histopathological changes with haemolymph immune assays may show whether the non-destructive haemolymph sampling can provide early warning of stressors that cause tissue damage. The methods used can be applied to scientifically assess any husbandry procedures with a view to improve stock management and productivity.

Introduction

Commercial land based abalone farming requires periodic movement of stock between tanks for such reasons as harvesting, grading into similar sizes and to reduce stocking density (Hooper et al., 2011). Abalone clamp their foot muscle tightly to the tank floor, making them hard to detach. They are removed either mechanically with a spatula (called chipping) or after applying anaesthetic to the tank (Hooper et al., 2011). It is not practical to shift entire tanks containing tens of thousands of stock via chipping, so anaesthetics are used for large scale movement procedures. Chipping is used to move abalone when they are near harvesting size to avoid the problem of residues in the meat. Manual removal can cause physical damage to the foot, leading to subsequent infection (Genade et al., 1988) and is labour intensive.

Many anaesthetics have been evaluated for the purpose of handling abalone, but these studies have concentrated on the effectiveness of the anaesthetic, recovery time, subsequent mortality and growth (Aquilina and Roberts, 2000, Bilbao et al., 2011, Burke et al., 2001, Sharma et al., 2003, White et al., 1996), rather than effects on physiology or tissue damage. Benzocaine dissolved in ethanol is commonly used by farmers to remove abalone from tanks, due to the effectiveness of the anaesthesia, rapid recovery and low mortality (Edwards et al., 2000) and is the anaesthetic method used on the farm where this research was carried out.

Benzocaine (ethyl p-aminobenzoic acid) is a local anaesthetic that works by blocking sodium channels (Pinto et al., 2005, Wang et al., 1997). Benzocaine, like other anaesthetics, leaves residues in the flesh with clearance efficiency dependent on factors such as salinity and water temperature (Chang et al., 2012, Stehly et al., 1998). Levels in fish decrease to below detectable levels within 24 h (Allen, 1988), but there are no equivalent studies in molluscs.

Previous investigation into the effects of benzocaine anaesthesia on abalone haemolymph indicated that the procedure results in acute depression of phagocytosis, antibacterial activity and lysosomal neutral red retention time, as well as elevated haemocyte density (Hooper et al., 2011). Observed effects were greatest when abalone were moved after having been anaesthetised, with lesser effects seen with anaesthesia alone. No significant differences were seen when abalone were removed from the tank by chipping when compared to the control group. Recovery generally occurred within one day for all treatments. This suggests a short period of vulnerability to pathogenic infection in abalone after anaesthesia and movement, due to immunosuppression. The potential for more chronic effects of anaesthesia and/or movement on organ systems or homeostasis also requires investigation.

There are only rare reports on the effect of anaesthetics on molluscan physiology. Nembutal anaesthesia in the gastropod Biomphalaria glabrata results in lower haemolymph glucose concentrations, with less variation in well-anaesthetised snails versus poorly-anaesthetised snails (Liebsch et al., 1978). The authors speculated that this difference was due to stress of handling on the poorly anaesthetised snails. Benzocaine induced a decline in metabolic rate in abalone (measured as oxygen consumption) that returned to normal after clearance of benzocaine (Chacon et al., 2003). Edwards et al. (2000) reported that chipping of abalone and benzocaine anaesthesia both caused suppression of oxygen uptake, with recovery one day after chipping and 3–5 days after benzocaine anaesthesia. Both chipped and anaesthetised abalone had lower growth rates than control animals, with no significant differences between these two treatments (Edwards et al., 2000). The results of this laboratory study by Edwards et al. (2000) suggest that, although the anaesthetic may have a more profound effect in the acute stage, in the long term it may not be more detrimental to abalone health than chipping. These studies were all done within the laboratory and the situation on farms may differ. The development of opportunistic infections is probably more likely on farms and our previous immune study (Hooper et al., 2011) suggests this is more likely with anaesthesia than with manual chipping. There are no published reports of the effect of benzocaine anaesthesia on histological changes in the tissues, haemolymph protein, nor ion concentrations in invertebrates.

Histopathology is used in disease investigation on abalone farms (Handlinger et al., 2006, Mouton, 2003) and can be used to characterise the lesions present as infectious (Hooper et al., 2007, Moore et al., 2000) or non-infectious (Elston, 1983, Harris et al., 1998). It is important to identify the range of background lesions that can occur in abalone tissues due to husbandry stressors such as anaesthesia and chipping, so that the background lesions associated with farming are not mistaken for lesions due to a disease outbreak. There are a few reports on the effect of on-farm husbandry stressors on abalone histopathology, but these are primarily descriptive rather than quantitative (Elston, 1983, Harris et al., 1998, Harris et al., 1999, Mouton, 2003).

The purpose of this investigation was both to examine the haemolymph biochemistry and quantify histological changes in abalone after anaesthesia and/or manual movement and to compare these with previous results on immune function changes (Hooper et al., 2011). Comparing the physiological and histopathological changes with haemolymph immune assays may show whether the non-destructive haemolymph sampling can provide early warning of stressors that cause tissue damage. One of the main purposes of this paper is to show farmers scientific methods to evaluate their current husbandry methods.

Section snippets

Animals and experimental design

The experimental design was as described in Hooper et al. (2011). Two year old hybrids of male Haliotis laevigata × female Haliotis rubra, weighing 30.1 g +/− SE 1.5, (N = 130), were housed in a shed containing 96 concrete flow-through slab tanks 20 m long and 1.8 m wide. Animals were held at a stocking density of 10,000 per tank (8.36 kg/m2) prior to movement. Three tanks were assigned for each treatment including controls and two abalone sampled from each tank per day. In three control tanks abalone

Biochemistry

ANOVA detected a significant day × treatment interaction (F6,57 = 3.04 p = 0.012) for haemolymph protein. The planned comparisons showed that the haemolymph protein was significantly elevated from day 1 to day 2 in both the chipped group and anaesthetised group compared to controls (Fig. 5a; Anaesthesia group F1,57 = 7.354, p = 0.009. Chipped group F1,57 = 7.216, p = 0.009). By day 3, recovery had apparently commenced in both treatment groups and by day 5, the recovery was significant in the anaesthetised

Discussion

Anaesthesia had a significant effect on abalone tissues irrespective of whether the abalone were subsequently moved. With most changes, recovery had occurred within one day. This is similar to the fast recovery in immunosuppression seen in these same abalone due to anaesthesia (Hooper et al., 2011).

Anaesthesia, with or without subsequent movement, damaged the foot epithelial cells in such a way that these cells started to slough within one day, whereas epithelial loss did not vary in the

Acknowledgements

We thank the Australian Government Fisheries Research and Development Corporation, which supplied the funding for this research; Great Southern Waters Limited for allowing the research to be carried out and supporting it on their abalone farm and Gribbles Veterinary Pathology for processing the tissue samples and haemolymph biochemistry.

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