Tobacco as Fish Sedative. Effect of Tobacco (Nicotiana tabacum) Leaf Dust as Sedative for Rohu, Labeo rohita (Hamilton, 1822) Fingerlings Transport
Master's Thesis 2016 111 Pages
2 REVIEW OF LITERATURE
2.1 Rohu in India
2.4 Stress during transport
2.5 Sedation and Anaesthesia
2.6 Characteristics of an ideal Anaesthetic
2.7 Sedatives and Anaesthetics in Aquaculture
2.8 Induction and Recovery Period
2.9 Behavioral Responses in Induction and Recovery Period
2.10.3 Active ingredients
2.10.5 Tobacco in India
2.11 Effect on water quality parameters
2.11.3 Dissolved oxygen
2.11.4 Dissolved free carbon dioxide
2.11.5 Total Alkalinity
2.11.6 Total hardness
2.12 Effect on haematological parameters
2.12.1 Total erythrocyte count
2.12.4 Red blood cell indices
2.12.5 Total leukocyte count
3 MATERIALS AND METHODS
3.1 Locus of Experiment
3.2 Experimental Fish
3.3 Experimental Design
3.4 Tobacco Leaf Dust Preparation
3.5 Experimental Setup
3.5.1 Sedation Dose for Transportation
3.5.2 Protocol for Simulated Transportation
3.7 Cleaning and Siphoning
3.8 Chemicals and Glass wares
3.9 Water Quality Analysis
3.9.3. Dissolved oxygen
3.9.4. Dissolved free carbon dioxide
3.9.5. Total Alkalinity
3.9.6. Total hardness
3.10. Haematological parameters
3.10.1. Blood collection
3.10.2. Total erythrocyte count
3.10.5. Total leukocyte count
3.10.6. Red blood cell indices
3.11. Statistical Analysis
It’s not only the efforts of me but also by the people who helped, inspired and motivated me. Without them it couldn’t be completed. So, it’s time to acknowledge the people from the deep of my heart with full of love and happiness. First and foremost, I want to praise and thank Jesus who showered grace on me and made everything possible for me to complete the dissertation work through provision of help by the people. I want to express my deep sense of gratitude wholeheartedly to my Appa, Amma, Machan, Akka, Kuttima and Sherry Abraham for the belief they had on me. They are the back bone for me providing great support, love and timely advice in all my situations. I love you all without you people I can’t do anything in my life.
I am extremely happy to say that I am greatly blessed to work under the guidance of Dr. Chandra Prakash, Principal Scientist, Division of Aquaculture, CIFE, Mumbai. Without his support it was unable to complete my dissertation. I am indebted for his way of approach, technical support, incessant supervision, valuable thoughts, generous attention and unobstructed kindness over me. A single word of thanks will not fulfil his immense and timely help. I am deeply overwhelmed to your parental care. Once again thanks a lot and I am grateful to you sir.
I thank my co-advisors, Dr. N.K. Chadha, Head & Principal Scientist, Division of Aquaculture and Dr. Nalini Poojary, Asst. Chief Technical Officer, Aquatic Environment and Health Management Division for their valuable support during indenting the chemicals, data analysis, supervision and recommendations given by them throughout my dissertation work.
I thank my lovable seniors Somu, Nirmal, Vicky and my besties GK, Siva and Sandal without whom it was impossible for me to complete my dissertation work. They were with me throughout all the happy and unhappy moments of my research work. I thank all my seniors especially Roshan Maria Peter, Shashank Singh, Sambid Swain, Arun VV, Nuzaiba, Lloyd, Dani and all seniors in CIFE and TNFU for their valuable suggestions, and I also thank my colleagues Abuthagir, Sri Hari, Velu, Ranjith, Madhu, Pranay, Gomathi.V, Gomathi.P, BharathiRathinam andJerusha.
No one say thanks to their sweethearts but here I have to mention their names Ezhil, Angel, Bavithra, Antony, Mari, Bavith, Raswin, Amala, Kavi and Petchi Muthu. I thank all my esteem batch mates for their contribution to my work especially Sanitha, Jess, Saikat, Vivek, Iffat, Siju, Shyam, Menaga and Nageswari.
I am very much thankful to all faculty and staff of the Division of Aquaculture for their support throughout my research work. Especially Dr. Neelam Saharan, Dr. Kiran Dube Rawat, Dr. A.K. Reddy, Dr. V.K. Tiwari, Dr. A.K. Verma, Dr. Paromita Benarjee Sawant and Dr. Babitha Rani A.M. for their valuable suggestions.
LIST OF TABLES
1. Commonly used Sedatives and Anesthetics in Aquaculture
2. Stages of anesthesia in fish
3. Stages of recovery from anesthesia in fish
4. Proximate content of commercial floating type pellet diet
5. Time (min) required for induction and recovery (stage 6) (Mean ± SE) of the anesthesia using tobacco leaf dust in L. rohita (Maximum observation time- 30 min)
6. Behavioural responses of rohu fingerlings to different doses of tobacco leaf dust at 2 h interval during 12 h exposure period in anesthetic bath
7. Mortality rate (%) (Mean ± SE) of rohu, L. rohita fingerlings found in polythene bags at different durations during simulated transportation experiment at 50g L-1 of loading density
8. Water temperature (0C) (Mean ± SE) observed at different time intervals during simulated transportation of L. rohita fingerlings
9. pH (Mean ± SE) observed at different time intervals during simulated transportation of L. rohita fingerlings
10. Dissolved oxygen (mg L-1) (Mean ± SE) observed at different time intervals during simulated transportation of L. rohita fingerlings
11. Dissolved free carbon dioxide (mg L-1) (Mean ± SE) observed at different time intervals during simulated transportation of L. rohita fingerlings
12. Total alkalinity (mg L-1) (Mean ± SE) observed at different time intervals during simulated transportation of L. rohita fingerlings
13. Total hardness (mg L-1) (Mean ± SE) observed at different time intervals during simulated transportation of L. rohita fingerlings
14. Ammonia (mg L-1) (Mean ± SE) observed at different time intervals during simulated transportation of L. rohita fingerlings
15. Nitrite (mg L-1) (Mean ± SE) observed at different time intervals during simulated transportation of L. rohita fingerlings
16. Nitrate (mg L-1) (Mean ± SE) observed at different time intervals during simulated transportation of L. rohita fingerlings
17. Phosphate (mg L-1) (Mean ± SE) observed at different time intervals during simulated transportation of L. rohita fingerlings
18. Total erythrocyte count (106 mm-3) (Mean ± SE) observed at different time intervals during simulated transportation of L. rohita fingerlings
19. Haemoglobin levels (g dL-1) (Mean ± SE) observed at different time intervals during simulated transportation of L. rohita fingerlings
20. Haematocrit value (%) (Mean ± SE) observed at different time intervals during simulated transportation of L. rohita fingerlings
21. Mean corpuscular volumes (fl) (Mean ± SE) observed at different time intervals during simulated transportation of L. rohita fingerlings
22. Mean Corpuscular Haemoglobin (pg) (Mean ± SE) observed at different time intervals during simulated transportation of L. rohita fingerlings
23 Mean Corpuscular Haemoglobin Concentration (g dL-1) (Mean ± SE) observed at different time intervals during simulated transportation of L.rohita fingerlings
24 Total leukocyte count (103mm-3) (Mean ± SE) observed at different time intervals during simulated transportation of L. rohita fingerlings
25 Lymphocytes (%) (Mean ± SE) observed at different time intervals during simulated transportation of L. rohita fingerlings
26 Monocytes (%) (Mean ± SE) observed at different time intervals during simulated transportation of L. rohita fingerlings
27 Neutrophils (%) (Mean ± SE) observed at different time intervals during simulated transportation of L. rohita fingerlings
28 Eosinophils (%) (Mean ± SE) observed at different time intervals during simulated transportation of Labeo rohita fingerlings
29 Basophils (%) (Mean ± SE) observed at different time intervals during simulated transportation of Labeo rohita fingerlings
30 Thrombocytes (104 mm-3) (Mean ± SE) observed at different durations during simulated fish transportation trial of L. rohita fingerlings
LIST OF FIGURES
1 Induction time of L. rohita fingerlings observed in anaesthetic bath during 30 mins observation time
2 Recovery time of L. rohita fingerlings observed in anaesthetic bath during 30 mins observation time
3 Mortality rate (%) of L. rohita fingerlings during the simulated transportation
4 Water temperature (0C) observed during simulated transportation of L. rohita fingerlings
5 pH observed during simulated transportation of L. rohita fingerlings
6 Dissolved oxygen (mg L-1) observed during simulated transportation of L. rohita fingerlings
7 Dissolved free carbon dioxide (mg L-1) observed during simulated transportation of L. rohita fingerlings
8 Total alkalinity (mg L-1) observed during simulated transportation of L. rohita fingerlings
9 Total hardness (mg L-1) observed during simulated transportation of L. rohita fingerlings
10 Ammonia (mg L-1) observed during simulated transportation of L. rohita fingerlings
11 Nitrite (mg L-1) observed during simulated transportation of L. rohita fingerlings
12 Nitrate (mg L-1) observed during simulated transportation of L. rohita fingerlings
13 Phosphate (mg L-1) observed during simulated transportation of L. rohita fingerlings
14 Total erythrocyte count (106 mm-3) observed during simulated transportation of L. rohita fingerlings
15 Haemoglobin (g dL-1) observed during simulated transportation of L. rohita fingerlings
16 Haematocrit (%) observed during simulated transportation of L. rohita fingerlings
17 Mean corpuscular volume (fl) observed during simulated transportation of L. rohita fingerlings
18 Mean Corpuscular Haemoglobin (pg) observed during simulated transportation of L. rohita fingerlings
19 Mean Corpuscular Haemoglobin Concentration (g dL-1) observed during simulated transportation of L. rohita fingerlings
20 Total leukocyte count (103 mm-3) observed during simulated transportation of L. rohita fingerlings
21 Lymphocytes (%) observed during simulated transportation of L. rohita fingerlings
22 Monocytes (%) observed during simulated transportation of L. rohita fingerlings
23 Neutrophils observed during simulated transportation of L. rohita fingerlings
24 Eosinophils (%) observed during simulated transportation of L. rohita fingerlings
25 Basophils (%) observed during simulated transportation of L. rohita fingerlings
26 Thrombocytes (104mm-3) observed during simulated fish transportation trial of L. rohita fingerlings
LIST OF PLATES
1 Dried Tobacco Leaves and Tobacco Leaf Dust
2 Length-Weight Measurement of Experimental Fishes
3 Sedative Efficacy Experiment
4 Simulated Transportation Experiment
5 Water Quality Analysis
6 Blood Collection
The current study was undertaken to investigate the effectiveness of tobacco leaf dust as sedative for the transport of rohu (Labeo rohita) fingerlings. The experiment of sedative efficacy and simulated transportation was conducted for 12 h in glass tanks (30 L capacity) and plastic bags (75 * 45 cm), respectively with different concentrations of tobacco leaf dust such as 0, 25, 50, 75, 100 and 125 mg L"1 among which 0 mg Ľ1 was used as control. The fingerlings (6.45 ± 0.68 cm and 3.29 ± 0.52 g) were stocked at a stocking density of 10 fishes / tank and 30 fishes / plastic bag in triplicates. The induction and recovery times observed in anaesthetic bath significantly got decreased and increased with increase in the concentrations of tobacco leaf dust. The lowest effective dose of tobacco that produced induction (< 15 min, preferably < 3 min) and recovery (<5 min) was 25 mg L'1. The tobacco leaf dust is effective in inducing light sedation in rohu at a concentration of 25 mg L'1. The mortality rate (15-40%) of fingerlings during transportation was significantly higher in the control (without sedative) than the sedative doses of tobacco. Poor water quality was noticed in control group with significant decrease in pH, DO and nitrate and significant increase in temperature, CO2, total alkalinity, ammonia, nitrite and phosphate. Serious changes were found in haemogram and leukogram of fingerlings in control group with significant decrease in RBC, Hb, Het, MCV, MCH, WBC, lymphocytes and thrombocytes while significant increase in MCHC, monocytes, neutrophils, eosinophils and basophils. Therefore, the present study indicates that L. rohita fingerlings can be successfully transported for up to 12 h using 25 mg L'1 tobacco leaf dust without any water quality deterioration in transport bags and haematologic alterations of fishes.
India is one of the richest nations in the world with regard to fish biodiversity, distributed over a wide network of freshwater ecosystems in the country. The nation accounts for 4.4% of the global fish production. Aquaculture in India is highly promising and has grown over the last two decades with freshwater aquaculture contributing over 95% of the total aquaculture production. The country shared 3.6 million tons to world freshwater fish production in 2009 (FAO, 2009). The aquaculture systems in India and its neighboring countries mainly constitute Indian major carps (Catla, Rohu and Mrigal). Rohu is a fast growing freshwater fish species which belongs to the family Cyprinidae and genus, Labeo. Among the three Indian major carps, rohu (Labeo rohita) is the species of significance preferred in poly-culture systems of carps because of its high growth potential. It is the most important species of great demand by the consumers owing to texture and taste of the carcass.
To maximize and sustain the production, healthy and quality seed is the most essential input. Fish seeds are produced in hatcheries and are supplied to the farms for grow-out culture. But many a times the hatcheries are located far away from the culture sites. Therefore, transportation of fish is one of the most significant operations in aquaculture which plays a crucial role in the supply of seeds from hatcheries to grow out farms. During transport, the metabolic activities of fish are three times more than that of the normal (Froese, 1998). Fishes undergo multi-phase of stress due to multiple stressors involved during transportation practices. Stress is an indispensable factor during the transportation of fishes. Rohu is a fish which is highly stressed as it is quite sensitive to the transport stress. Consequently, there is a high mortality rate (about 90%) among transported rohu fingerlings (Lewis et al., 1996), since packing them at high densities get resulted in confinement (Husen and Sharma, 2015). Transport stress in fishes can be minimized with the help of light sedation, i.e., low concentration of an anaesthetic (Ackerman et al., 2005). Therefore, in order to reduce the stress induced mortality, natural or synthetic sedatives and anesthetics can be used to sedate and immobilize fish (King et al., 2005; Ross and Ross, 1999).
Sedation is a mild form of anesthesia which puts the fish to sleep by calming and immobilizing their activities. The application of sedatives silences the activity of fishes and is beneficial in lowering the stress induced mortality during transportation (Strange and Schreck, 1978). Sedative and anesthetic substances used for fishes must have: rapid induction and recuperation period, safe margin to fish and humans, no physiological and residual effect, local availability and low cost (Marking and Meyer, 1985). Several anaesthetics have been evaluated and used so far. Each one has its own merits and demerits. Some are toxic and inexpensive while some are expensive and less toxic. As of now, MS-222 is the only anaesthetic that has a USFDA approval to be used as an anaesthetic in food fishes irrespective of its demerits like low pH, low efficacy on plasma cortisol control and expensiveness (Coyle et al., 2004). To avoid adverse stress effects from anaesthetics, knowledge on optimum concentration of an anaesthetic for different fish species is more significant (Hoseini era/., 2011; Hoseini and Ghelichpour, 2012). For practical use in aquaculture, only few studies have been conducted in commonly cultured tropical freshwater food fishes (Husen and Sharma, 2014).
Tobacco, Nicotiana tabacum, is a herbaceous annual or perennial plant in the family Solanaceae (Night shade), grown for its leaves. It is a medicinal plant of remarkable benefits and history of use in traditional Indian medicine as a sedative, antispasmodic, and vermifuge with high alkaloid content mainly nicotine and it is under the utmost care of humans. It has a potential to heal and protect when used effectively but has the ability to harm when abused (Binorkar et al., 2012). India stands 3rd in tobacco production in the world next to Brazil and USA (Tobacco Board, 2016). The expensive anaesthetics are scarce in developing and under developed countries whereas inexpensive anaesthetics are toxic and harmful to fish and humans with more deleterious effects. Therefore, in search of safe, effective and less expensive sedatives and anaesthetics, tobacco which is of natural origin, cheap, easily available and eco-friendly narcotic could be a novel and futuristic sedative and anaesthetic for fishes during transport and surgeries.
Only limited work is done in Africa using tobacco as piscicide and as an anaesthetic. Therefore, a study on, effect of tobacco (Nicotiana tabacum) leaf dust as sedative for rohu, Labeo rohita (Hamilton, 1822) fingerlings transport was proposed with the following objectives.
1. To determine the efficacy and optimum concentration of tobacco as sedative on Labeo rohita fingerlings.
2. To evaluate the haematological response of Labeo rohita exposed to different concentrations of tobacco.
2. REVIEW OF LITERATURE
2.1. Rohu in India
Rohu is an indigenous food fish of India that belongs to the family cyprinidae genus Labeo. It is one of the most preferred species in the country fetching a higher price in the market among the Indian major carps. It is cultured in many parts of the country and other adjacent countries in South East Asia owing to its high growth potential coupled with high consumer preference. It is the preferred species of importance in the polyculture systems (FAO, 2001).
Stress is defined as a set of physiological reactions to one or more stimuli that threaten homeostasis. Fishes, with a series of neuro-endocrine manipulations respond to one or more stressors known as the stress response (Silva et al., 2012). There are three types of response to stress, namely primary, secondary and tertiary. In primary response to stress the two major systems: the hypothalamic- pituitary-interrenal (HPI) axis and the sympathetico- chromaffin (SC) system are activated. This stimulation of the HPI axis and SC system results in increased circulating levels of cortisol and adrenaline respectively. Secondary responses in turn are stimulated by the above neuroendocrine reactions in which they are reflected as changes in biochemical, physiological, haematological and immunological parameters (Barton and Iwama, 1991). Tertiary responses follow when the stress is severe or prolonged and these include the modifications in disease resistance, growth, reproductive output and overall condition and market quality (Lowe era/., 1993; Sumpter, 1991). In aquaculture practices, stress is an unavoidable component which is always associated with netting, handling, transportation, water and sediment quality, vaccination and disease treatment (Pakhira et al., 2015). Jensen (1990) reported that stress causes adverse effects which are additive and cumulative. It can also result in immune suppression, physical injury, stunted growth and even death (Husen and Sharma, 2015).
The hatcheries are many a times located far way from the culture sites, therefore, transportation of fish is one of the most significant operations in aquaculture as it plays a vital role in the supply of seed from hatcheries to grow out farms. It is an operation which is inexorable and indispensable to aquaculture. In numerous cases, to stock in ponds, fry and fingerlings have to be transported from hatcheries. The traditional method of transport is open containers and splashing with hands or legs to get mixed oxygen into the water. Modern methods of transport have done away with open containers, which are voluminous and heavy and the need for labours, water exchange and agitation for oxygenation.
Live-fish transport in polyethylene bags is the modern method and is now common in the field of trade as it imparts the reduction in the shipping weight of fish consignments facilitating transport by airways (Lim et al., 2003). The seed are packed at higher stocking densities in these plastic bags with 1/3 water and 2/3 oxygen. Prior to handling and transport, the fishes should be conditioned in well aerated water without feeding for two or three days as it not only reduces the respiratory stress but also prevents the deterioration of water quality caused due to faecal material and regurgitated food (Steele et al., 2010). The transportation of fishes may be of different durations depending on the distance between the target regions and the hatchery sites. The duration of transport is divided into short term (< 8h) or long term (> 8h) with the threshold of 8h as the distinction between short and long transport (Stieglitz et al., 2012).
2.4. Stress during transport
Fishes undergo multi-phase of stress due to multiple stressors involved during transportation practices. With several potential stressors, such as capture, loading, transport, unloading, temperature differences, water quality deterioration and stocking, transport is considered as one of the most stressful procedures in aquaculture practices (Inoue et al., 2005; Husen and Sharma, 2015). The fishes detect these stressors and react to them by triggering a series of physiological reactions through two pathways, the BPI (brain-pituitary-inter-renal) axis or the BSC (brain-sympathetic-chromaffin cell) axis through their nervous system (Boijink et al., 2016). This response ultimately brings changes in plasma cortisol, glucose, lactase, plasma chloride, sodium, and lymphocyte concentrations in fish (Husen and Sharma, 2014).
During transport, the metabolism offish is three fold higher than that of the normal (Froese, 1998). Packing of fishes at higher densities in a small volume of water is the current practice in the industry to reduce the weight and thereby the shipping costs (Guo et al., 1995). This practice deteriorates the quality of water, leading in low dissolved oxygen (DO) and high carbon dioxide content and thus rapid accumulation of metabolic wastes (Teo et al., 1989; Froese 1998), which can cause for the mortality of fishes categorized as either dead on arrival (DOA) or dead after arrival (Correia, 2001; Lim et al., 2003). Lewis et al. (1996) reported that the mortality resulting from transport is as high as 90% and is common. Osmoregulatory disfunction or stress-mediated diseases are the causative agents for fish mortalities during and after transportation events (Ims, 2011). Fish transportation, in general, is conducted in crowded conditions with higher stocking densities which deteriorate water quality as well as the health status of fishes after transport (Charoendat et al., 2009).
Rohu is quite sensitive to the transport stress and, consequently, there is a huge mortality among transported rohu fingerlings (Lewis et al., 1996). Packing them in small quantity water at high densities results in confinement which are stressful to rohu fingerlings during transport (Husen and Sharma, 2015) that ultimately leads to mortality.
Some authors have reported that stress can also occur due to transport duration. Das et al. (2015) suggested that the transport duration and the physico-chemical parameters of the water media determines the severity of transport stress. Noga (2000) also reported the same, indicating the fact that duration of transport influences the stress impact. However, in general, it is impractical to significantly reduce the transport duration in most cases (Sampaio and Freire, 2016). Short and long duration transports, both can be stressful to fish but it can be minimized through the application of sedatives or anaesthetics and some have been used in this respect (Altun and Dañabas, 2006). Light sedation which is brought about by low concentrations of an anaesthetic helps in minimizing the stress due to transport (Ackerman et al., 2005). Fish transportation without anesthetics might possibly uplift the ammonia level through excretion of metabolites, which, due to long-term exposure affects their gills (Balamurugan et al., 2016).
2.5. Sedation and Anaesthesia
Sedation is defined as a mild form or preliminary state of anaesthesia. It induces calming effect and drowsiness in fishes with dulled sensory perception and perhaps with some in-sensibilities to pain (analgesia) but with no gross loss of sensory perception or of equilibrium and is convenient for small non-invasive/non-painful procedures such as handling, weighing, simple marking and imaging (Ross and Ross, 2008). To mitigate stress and to increase fish welfare, sedation is an important and useful procedure (Ashley, 2007; Southgate, 2008). Bauquier et al. (2013) suggested that during procedures such as physical examination, diagnostic sampling or transport, sedation will minimize the stress and injury risk experienced by the fish.
Anaesthesia is generally defined as a state of loss of sensation or insensibility caused through nervous system depression when an external agent is applied (Ackerman et al., 2005; Ross and Ross, 2008) and can be introduced to fish through physical or chemical techniques. It is a procedure of significance in aquaculture that results in better and safer management practices during biometry, blood collection and body inspections because of partial and facultative immobilization of fishes (Boijink et al., 2016). Anaesthetic conditions not only lessen the stressor perception of the fishes but also prevent their nerve signal transmission to the central nervous system (Woods et al., 2008). Silva et al. (2012) observed the infection in fishes as well as their appetite inhibition and concluded that it could be the result of concomitant stress from handling or confinement stress in addition to anaesthesia. Although fish physiology can be altered by anaesthetic treatments, light anesthesia can have silencing effect on fish and lowers the stress caused during handling and transport (Steele et al., 2010).
2.6. Characteristics of an ideal Anesthetic (Marking and Meyer, 1985)
The anesthetic agent should have, an induction time of less than 15 minutes and preferably less than 3 minutes and recovery of less than 5 minutes, no toxic effects to fish and humans with relative safety margin, no negative effects on fish physiology leaving no residues, no repeated exposure problems and low cost.
2.7. Sedatives and Anaesthetics in Aquaculture
Sedatives and anaesthetics are useful tools that play a significant role in carrying out many operations such as blood sampling, tissue collection, surgery, handling and transport since they partially or completely immobilize the fish restricting their physical activity. Rapid increase of intensive aquaculture as a result of high demand for fish made aquaculture more intensive which has led to the demand and application of novel chemicals (Kanani et al., 2011). Strange and Schreck (1978) observed that the application of sedatives reduce physical activity and stress of fish during handling and may be beneficial in fish transportation to reduce mortality and to overcome the multiple stressors. Anaesthetics help in slowing down the metabolic rate of fish and thereby reducing oxygen uptake and decreasing the buildup of carbon dioxide and ammonia during transport (Harmon, 2009). It also reduces the stress perception and responsiveness creating a depression in nervous function thereby minimizing injury and mortality of the fishes during transport (Iwama and Ackerman, 1994; Ims, 2011).
The most common anaesthetic technique in fish is immersion anaesthesia, i.e., adding the anaesthetic agent to the water. In this technique, the fishes are exposed to anaesthetic bath treatments which are similar to inhalation anaesthesia in human and veterinary medicine (Zahl et al., 2009). Sevaral anaesthetics have been evaluated and some are used for aquaculture applications so far (Marking and Meyer, 1985; Gilderhus and Marking, 1987; Summerfelt and Smith, 1990; Stoskopf, 1993; Ross and Ross, 1999). Among them, tricaine methanesulphonate (MS-222), 2-phenoxyethanol, quinaldine, benzocaine and metomidate are the major ones (Mylonas et al., 2005). Coyle et al. (2004) reported that MS-222 is the only anaesthetic that has a USFDA approval to be used as an anaesthetic in food fishes irrespective of its demerits like low pH, low efficacy on plasma cortisol control and expensiveness. MS-222 is almost 100 times that of quinaldine in cost (Hseu et al., 1998). Some sedatives and anaesthetics are expensive and some are inexpensive but each one has its own merits and demerits.
In addition to cost, toxicity (to fish and users), efficacy, withdrawal time in experimental species, etc. are the criteria which have to be considered before selecting a suitable anaesthetic (Summerfelt and Smith, 1990; Hseu et al., 1998). It must be taken into consideration that anaestheics or sedatives used should not cause stress to the fish when introduced (Hill and Forster, 2004). Reports envisage that anaesthetics can also affect fish by alleviating stress in them (Davis era/., 1982). Ims (2011) has also observed that abnormal effects may occur due to some anaesthetic drugs and improper dosages. Therefore it is more important and essential to discover a suitable anaesthetic with an appropriate dosage (Carter et al., 2010).
Table 01: Commonly used Sedatives and Anaesthetics in Aquaculture (Iwama and Ackerman, 1994; Stoskopf, 1993).
Abbildung in dieser Leseprobe nicht enthalten
2.8. Induction and Recovery Period
Fish exposed to anaesthetic undergo three phases, viz., induction, maintenance and recovery (McFarland and Klontz, 1969; Ross and Ross, 2008). The period of induction is the time from the moment the fishes are exposed to anaesthetic until the total loss of equilibrium, i.e., slow but regular respiratory movement (Jolly et al., 1972). It should last for a few minutes but too rapid induction may stress and kill the fish. The preferable induction time is within 3 minutes (Marking and Meyer, 1985). The term maintenance is rarely used as only induction and recovery are clearly seen in anaesthetized fishes. Maintenance can be visible in sedated fishes and it is known as maintaining the fishes in calm mode putting them to sleep. This can be achieved with a help of light sedation which is essential for maintenance during transport that minimizes stress and mortality. The period of recovery is the time from the moment the anaesthetized fishes take to regain its equilibrium until it attains the full recovery (Tsantilas et al., 2006). It should be rapid to mitigate the harmful effects on fish (Woods et al., 2008). The preferable recovery time is within 5 minutes (Marking and Meyer, 1985). It has an inverse relationship with dosage and exposure time (McFarland and Klontz, 1969).
2.9. Behavioural Responses in Induction and Recovery Period
Fishes exhibit a different sign of behaviour during induction and recovery. Based on behavioural signs, the sedative and anaesthetic effects have been classified into different stages by McFarland (1959) and he was the first to do it. Neiffer and Stamper (2009) reported that the induction phase is generally expressed by decrease in caudal fin strokes, swimming and respiratory activity and response to stimuli; low caudal fin stroke activity is usually the first sign, followed by loss of equilibrium and response to stimuli whereas recovery phase showed variability in behaviour indicating the increase in respiration, muscle tone return, fin movements resume, and swimming activity with progressively less ataxia to regain its full equilibrium.
Table 02: Stages of anesthesia in fish (Keene et al., 1998, modified from McFarland, 1959; Jolly et al., 1972).
Abbildung in dieser Leseprobe nicht enthalten
Table 03: Stages of recovery from anesthesia in fish (Keene et al., 1998 modified from Hikasa et al., 1986).
Abbildung in dieser Leseprobe nicht enthalten
Linnaeus derived the genus Nicotiana using French diplomat’s name Jean Nicotine de Villeman. He is the one who introduced tobacco plant in France in 1560 from Brazil for the treatment of different ailments which was further spread to rest parts of Europe. Tobacco, Nicotiana tabacum, is a herbaceous annual or perennial plant of the family Solanaceae (Night shade) grown for its leaves.
The tobacco plant has a thick, hairy stem and large, simple leaves which are oval in shape. The tobacco plant produces white, cream, pink or red flowers which grow in large clusters, are tubular in appearance and can reach 3.5-5.5 cm (1.25- 2 inch) in length. Tobacco may reach 1.2-1.8 m (4-6 ft) in height and as is usually grown as an annual, surviving only one growing season (www.plantvillage.org).
Binorkar et al. (2012) reported that Nicotiana tabacum, the plant which has now been raised for commercial tobacco production, is probably of South American origin and Nicotiana rustica, the other major species which was carried around the world, came from North America. Both species are found distributed from Florida to New Mexico, to Massachusetts, New York, Southern Ontario and Minnesota.
2.10.3. Active ingredients
Tobacco is commonly considered as narcotic and encompass many uses as pesticide, piscicide, molluscicide and anaesthetic since it contains many phytochemicals like nicotine, anabasine (an alkaloid similar to the nicotine but less active), glucosides (tabacinine, tabacine), 2,3,6-trimethyl 1,4-naphthoquinone, 2-methylquinone, 2-napthylamine, propionic acid, anatalline, anthalin, anethole, acrolein, anatabine, cembrene, choline, nicotelline, nicotianine and pyrene. (Aleem, 1983; Agbon et al., 2002). Among the phytochemicals, nicotine is the most important and active ingredient which contributes between 2-5% dry weight of leaves (Hassal, 1982; Adamu, 2009). Pyridine and pyrrolidine ring constitute the nicotine (C5H4N)- CH-(CH2)3-N-(CH3). It is found more in roots and are transported to the leaves for storage. It is a volatile inflammable oil which is highly alkaline, with an acrid smell and a burning taste. Nicotine is readily soluble in water and in other non-polar solvents like alcohol, chloroform, ether and kerosene (Vogue, 1984). Konar (1977) reported that the toxic effect of nicotine was nil in 8 days and in 30 days for alkaline and neutral waters respectively.
Aleem (1988) suggested that tobacco leaf dust can be a used as a sedative and anaesthetic due to its low cost, local availability and complete degradability. The active ingredient nicotine is lipophilic in nature which can be easily absorbed by body and also can penetrate the epithelial and blood cells since it is highly soluble in membrane lipids (Hassal, 1982; Dani et al., 2001). Musa et al. (2013) reported that the toxicity of tobacco is negligible within three days after introduction indicating the fact that tobacco is bio-degradable and environmentally safe. Wan et al. (1996) have also pointed out that tobacco and tobacco based products are less harmful to fishes. There are eight criteria used to define an ideal anesthetic (Marking and Meyer, 1985). Tobacco is a much effective plant meeting five of the eight criteria with the favourable traits such as low cost, local availability, biodegradability, relative safety to humans and fish since being a natural product (Agokei and Adebisi, 2010). Therefore, cheap and eco-friendly tobacco could be a novel and better alternative to costly and toxic synthetic sedatives and anaesthetics (Jegede, 2014).
2.10.5. Tobacco in India
Next to Brazil and USA, India stands 3rd in tobacco production and exports with an annual production of about 800 million kg earning 20,000 crores approx by way of excise duty to the national exchequer, and 5000 crores approx by way of foreign exchange every year (Tobacco Board, 2016). In India, around 0.25% of cultivated land is used for the production of tobacco (Binorkar et al., 2012). Among the 29 states in the country, Andhra Pradesh, Gujarat, Karnataka and UP alone together account for over 90% of the total tobacco production in India. Tobacco is also grown in other states like Bihar, Maharashtra, Orissa, Tamil Nadu, and West Bengal (Goyal era/., 2004).
2.11. Effect on water quality parameters
Water quality deterioration occurs rapidly when the fishes are under crowding and hyperactivity conditions. This in turn leads to stress or even death of the fishes due to spontaneous changes in pH, hardness, alkalinity, temperature and other variables (Jensen, 1990). These conditions can be minimized through light sedation or anaesthesia. Both the anaesthetics and water quality parameters are inter-dependent, i.e., application of anaesthetics can affect water quality parameters and vice versa (Ims, 2011). Waters, wherein the fishes live are recommended to use for anaesthetic treatments in order to avoid the unendurable effects on the fish. Burka et al. (1997) observed that environmental factors like temperature, pH, salinity, chemical additives and dissolved oxygen have effect on the chemical properties of the anaesthetics. In the same way, optimum concentrations of anaesthetics are also influenced by species, water temperature, and other ambient physical and chemical characteristics (Hikasa et al., 1986; Son et al., 2001).
Temperature is defined as the degree of hotness or coldness in the body of a living organism. It is a critical parameter which influences other water quality parameters (Jensen, 1990). It influences DO concentrations in addition to its effects on metabolism, and can aggravate an anesthetic-induced hypoxia as high temperature leads to depletion of DO (Neiffer and Stamper, 2009). Temperature also affects induction and recovery period of anaesthetic drugs. Bowker et al. (2014) found that rapid induction and recoveries were associated with warmer temperatures. In the same way, Kristan et al. (2014) concluded that low temperature prolonged induction and recovery times. Temperature will also affect perfusion of gills affecting the uptake of anesthetic and its excretion (Ross and Ross, 2008; Burka et al., 1997). Therefore, it would be appropriate to provide special care to water temperature which may influence the metabolism of anaesthetic drugs in fish (Guénette et al., 2007). The suitable water temperature for transporting tropical fishes ranges from 18-28°C (Jhingran and Pullin, 1985).
It is the negative logarithm of hydrogen ion concentration in water. Ideal range of pH for fish culture is 7- 8.5. Carbon dioxide greatly influences the pH of water (Boyd, 1979). The suitable pH for hauling water is 7-7.5 and higher pH is the reason for uplifting the toxicity of unionized ammonia (Jensen, 1990). Reduction in water pH during transport occurs due to carbonic acid buildup resulting from carbon dioxide release during fish respiration (Boyd 1982; Swann, 1992). Jegede and Olanrewaju (2012) suggested that production of acidic metabolites, the decline in pH with increase in exposure time is due to higher solubility of CO2 in water. Ross and Ross (1984) observed that a drop in pH reduced the absorption of immersion anaesthetic solution due to increased ionization concluding that pH has influence the efficacy of anaesthetic.
2.11.3. Dissolved oxygen
Oxygen in its molecular state O2 is essential for many metabolic processes that are vital to aerobic life and aerobic organisms cannot exist without oxygen, which nevertheless is inherently dangerous to their lives. Due to low solubility of oxygen in water, obtaining sufficient oxygen is a greater problem for aquatic organisms than terrestrial ones and solubility also decrease with increase in factors like temperature and salinity (Bhatnagarand Devi, 2013). In fish transport, dissolved oxygen is an important limiting factor (Jensen, 1990). The transportation offish in plastic bags has limitations in oxygen supply giving rise to the build-up of ammonia and carbon dioxide levels (Becker era/., 2012, 2013; Carneiro et al., 2009; Golombieski et al., 2003). High mortality is noticed during transportation of live fishes due to lack of adequate oxygen present in the water (Jhingaran, 1975). Anaesthetics also cause oxygen debt but it is comparatively lesser than that caused by the handling (Hill and Forster, 2004).
Poor DO levels lead to more fish mortality, either directly or indirectly (Bhatnagar and Garg, 2000). According to Bhatnagar and Singh (2010) and Bhatnagar et al. (2004) DO level >5ppm is essential to support good fish production.
2.11.4. Dissolved free carbon dioxide
Free carbon dioxide is the main source of carbon path way in the nature and is contributed by the respiratory activity of animals. It is a highly soluble gas which when dissolved in water readily forms carbonic acid which decreases the pH of any system and can be harmful for aquatic organisms. But high oxygen levels can minimize the adverse effects resulting from carbon dioxide buildup (Jensen, 1990). Oxygen-carrying capacity offish blood is reduced when carbon dioxide exceeds above 20 mg/L (Berka 1986; Swann 1992). Swann (1997) suggested that a concentration of 10 ppm can be tolerated by fishes provided the DO concentrations are high. According to Bhatnagar et al. (2004), 5-8 ppm is essential for photosynthesis; 12-15 ppm is sublethal and 50-60 ppm is lethal to fishes.
2.11.5. Total Alkalinity
Alkalinity is the water’s ability to resist changes in pH and is a measure of the total concentration of bases in water including carbonates, bicarbonates, hydroxides, phosphates and borates, dissolved calcium, magnesium and other compounds in the water. Alkalinity (НСОз) results from the reaction of hydroxyl ion (produced when ammonia released by fish reacts with water) with CO2 (Boyd, 1990). Das et al. (2015) reported that the total alkalinity plays a significant role in carp seed survival and water with alkalinity above 100 mg СаСОз L'1 is usually considered unfavourable for IMC fry.
2.11.6. Total hardness
Hardness is the measure of alkaline earth elements such as calcium and magnesium (divalent metallic ions) in an aquatic body along with other ions such as ferrous, manganese, strontium, zinc, and hydrogen ions. The acceptable range of hardness in aquaculture is 50-150 ppm. Jensen (1990) reported that handling and transporting fish in soft waters (<10 ppm hardness) results in weak fishes or chronic fish losses and concluding that 50-100 ppm hardness and alkalinity are preferable.
Ammonia is the by-product from protein metabolism excreted by fish and bacterial decomposition of organic matter such as wasted food, faeces, dead plankton, sewage etc. The unionized form of ammonia (NH3) is extremely toxic while the ionized form (NH4+) is non-toxic. Both the forms are grouped together as “total ammonia”. It is main waste product excreted by fish after protein metabolism with toxic un-ionized ammonia in hauling water and the same is the case for fish transportation in closed systems (transport bags) (Berka,1986; Pramod et al., 2010). Ammonia cannot be removed by water agitation rather by using fishes with empty stomach and clean water with low temperature (Jensen, 1990). Bhatnagar et al. (2004) suggested that a concentration of 0.01-0.5 ppm is desirable for fishes and shrimps. However, critical concentrations of toxic ammonia are scarcely obtained under standard fish transport conditions.
Nitrite is an intermediate product of the aerobic nitrification bacterial process, produced by the autotrophic Nitrosomonas bacteria combining oxygen and ammonia. A rise in NO2 concentration in water is considered noxious to fish (Das et al., 2015). Nitrogenous compounds (e.g.,ammonia and nitrite) affect the uptake and excretion of immersion/inhalent anesthetics by modifying the gill morphology and can have several impacts on the metabolism of anaesthetic drugs. Methemoglobinemia reduces the oxygen-carrying capacity of blood which results from the marked elevation of nitrite (Neiffer and Stamper, 2009). The ideal and normal measurement of nitrite is zero in any aquatic system. According to Bhatnagar et al. (2004), a concentration between 0.02-1.0 ppm is lethal to many fish species.
Nitrate is harmless and is produced by the autotrophic Nitrobacter bacteria combining oxygen and nitrite. Nitrate levels are normally stabilized in the 50-100 ppm range. According to Stone and Thomforde (2004), nitrate is relatively non-toxic to fish but considered toxic only when the levels exceed above 90 mg Ľ1. The desirable range of nitrate for fishes is 0.1 - 4.5 ppm (Bhatnagar and Devi, 2013).
Almost all of the phosphorus (P) present in water is in the form of phosphate (PO4). It is an essential plant nutrient as it is often in limited supply and stimulates algal growth and its role for increasing the aquatic productivity is well recognized. Bhatnagar et al. (2004) suggested that a concentration between 0.05-0.07 ppm is optimum and productive; 1.0 ppm is good for plankton production.
2.12. Effect on haematological parameters
Haematological parameters are important tools that can be used as an effective and sensitive index to monitor physiological and pathological changes in fishes and have proven valuable for the quick detection of changes in fish health (Erhunmwunse and Ainerua, 2013; Nikoo et al., 2012; Kavitha et al., 2012). Blood parameters truly reflect physical and chemical changes occurring in organism. Therefore, studying the blood parameters provide a detailed information on general metabolism and physiological status offish of different age groups and habitat. Vázquez and Guerrero (2007) reported that transportation can affect haematological parameters. Hoseini et al. (2011) demonstrated that blood parameters particularly stress indicators are affected by anaesthetic concentrations and their exposure period. Results of several studies also suggest that the changes in the haematology and biochemistry of exposed fish can affect physiological conditions of the fish (Javahery and Moradlu, 2012). Therefore, it is important to evaluate blood parameters to find the physiological changes in anaesthetized fishes (Inoue et al., 2005; Park et al., 2008).
2.12.1. Total erythrocyte count
The erythrocyte count (RBC) measures number of red blood cells present in the blood. They are the dominant cell type in the blood of the vast majority of fish species. Witeska et al. (2015) found an increase in RBC of carp during deep anaesthesia with 2-phenoxyethanol and etomidate. RBC, haemoglobin and haematocrit are often elevated during stress situations to increase oxygen carrying capacity and oxygen supply to the major organs in response to higher metabolic demands (Ruane et al., 1999). Dobsikova et al. (2006) reported that haematological investigations are equivocal. Therefore, stress condition also leads to decrease in Hb content that may indicate a decrease in the rate of Hb synthesis which leads to impaired oxygen supply to various tissues resulting decrease in the number of RBC through haemolysis (Larsson et al., 1985).
Haemoglobin is a sophisticated oxygen delivery system that provides the desired amount of oxygen to the tissues under a wide variety of circumstances (Voet and Voet, 1990). It is a key parameter to point out the environmental conditions and it could be used as biomarkers in evaluating the environmental stress on fish as well as the changes in water quality (Kulkarni, 2015). Frisch and Anderson (2000) reported that stress associated with handling and transport evoked significant changes in haemoglobin levels of coral trout (Plectropomus leopardus).
Packed cell volume is a measure of the cellular fraction of erythrocytes and is a common indicator of stress in fish (Barton, 2002). Radoslav et al. (2013) suggested that the changes in PCV values can be used as a reliable indicator for determination of oxygen demand of the organisms. In Albula vulpes, Murchie et al. (2009) found out that stress caused by relocation process elicited secondary stress responses at haematological levels by altering PCV level.
2.12.4. Red blood cell indices
RBC indices helps to diagnose the cause of anaemia. Mean corpuscular volume (MCV) is the average size of red blood cell, mean corpuscular haemoglobin is the haemoglobin amount per red blood cell and mean corpuscular haemoglobin concentration is the amount of haemoglobin relative to the size of the cell (haemoglobin concentration) per red blood cell. Radoslav et al. (2013) suggested that decrease in MCHC values can be related to increase in erythrocyte volume (MCV) but not to increase of haemoglobin in them (MCH). Kavitha et al. (2012) found a decrease in MCHC values with increase in MCV and MCH values associated with toxic stress in fish.
2.12.5. Total leukocyte count
Leukocytes play a vital role in defence mechanism of fish. It is differentiated into granulocytes (neutrophils, eosinophils and basophils) and agranulocytes (lymphocytes and monocytes). Leukocyte profiles are particularly useful in the field of conservation physiology as they are altered by stress and can be directly related to stress hormone levels (Davis et al., 2008). Stress causes inconsistency in WBC and differential leukocyte count (Hoseini and Ghelichpour, 2012). Lymphocytes and granulocytes are important immune components and their reduced levels are commonly associated with live fish transport which leads to the increase in susceptibility of diseases (Frisch and Anderson, 2000). Stoskopf (1993) also reported that shift in leukocyte indices is considered an appropriate indicator in fish associated with stress response.
Thrombocytes are the most abundant cells next to erythrocytes. They are an important component of the blood clotting mechanism in fish (Houston, 1990). Ellsaesser and Clem (1986) found a significant reduction in peripheral blood thrombocytes after handling and transport stress, and Pickering and Pöttinger (1987) also found the reduction in the proportion of circulating thrombocytes due to chronic stress.
3. MATERIAL AND METHODS
3.1. Locus of Experiment
Field trials and Lab experiments were conducted at the wet laboratory unit and biology laboratory unit of Division of Aquaculture, ICAR- Central Institute of Fisheries Education, Andheri (West), Mumbai, India.
3.2. Experimental Fish
Labeo rohita fingerlings of 6.46 ± 0.68 cm mean length and 3.29 ± 0.52 g mean weight were procured from Maharastra State Fish Seed Farm, Aarey, Goregaon, Mumbai and the same were used as experimental fish. The fish were disinfected with 5 ppm КМпОд and were acclimatized in 300 L capacity FRP tanks at the wet laboratory unit, Division of Aquaculture, ICAR- Central Institute of Fisheries Education, Andheri (West), Mumbai, India.
3.3. Experimental Design
A completely randomized design was used to carryout the experiment. The experiment was conducted with triplicates for both the transit times of five treatments and the same was followed for the control as well.
3.4. Tobacco Leaf Dust Preparation
Good quality tobacco leaves were procured from a retailer in Kerala. The leaves were sun-dried for 7 days and were ground into fine dust (powder) with the help of mixer. The fine ground tobacco leaf dust was then stored in an air-tight container and used for the experiment.
3.5. Experimental Setup
300L capacity cylindrical FRP tanks of (5 No.) were taken and disinfected with bleaching powder (CaOCb) solution. Further, the tanks were rinsed and washed with freshwater and then sun dried. The 1000 L. rohita fingerlings were stocked in 5 FRP tanks @ 200 nos / tank. The fishes were maintained in these tanks for 15 days prior to the experiment of transportation.
3.5.1. Sedation Dose for Transportation
Five doses of tobacco leaf dust ( 25 mg L'1, 50 mg L'1, 75 mg L'1, 100 mg L"1, 125 mg L'1 ) as predetermined earlier were tested for sedative potential on L.