Use of Salt, Anesthetics, and Stocking Density in Transport of Live Fish: A Review

: For freshwater or marine fish farming to be successful, live fish must be transported. This can be performed with eggs, larvae, juveniles, and adults. Nonetheless, because of the handling, confinement, and duration of transport, it is considered a difficult procedure. To prevent significant physiological changes that could result in fish mortality, it is crucial to look for ways to reduce stress and enhance the quality of transport water. Consequently, a comprehensive range of research on the use of additives in water, including salt and artificial or natural anesthetics, is presented in this review, which also discusses potential changes in water quality factors during transport, such as dissolved oxygen (DO), carbon dioxide (CO 2 ), pH, ammonia, and temperature. The usage of stocking densities during transport is also covered in this review, with consideration given to the various recommendations for each species, fish size, and length of transport.


Introduction
The transport of live fish is receiving a high degree of attention because it is a fundamental step for successful fish production and research.Transport may be necessary for forming the breeding stock of a fish farm through the capture of wild breeders [1], as well as within the fish farm itself for reproduction control or between fish farms.Transport can also be carried out with eggs [2,3] and larvae [4,5].The sale of fish is totally dependent on efficient and safe transport for these animals since most fish farms do not carry out the entire production cycle in the same location.With the growing demand for sport fishing, it has also become increasingly necessary to transport larger fish [6,7].However, if this procedure is carried out unprofessionally, it can impact the well-being of the fish, causing stress, greater susceptibility to disease, and mortality, which may result in delays in the production cycle and serious economic losses for the producer.
The duration of transport can be short (2 h) [8] or long (72 h) [9], and generally, the most common type of transport is carried out in trucks or cars [10][11][12][13], although it can also be carried out using airplanes [13] or boats [14][15][16][17][18][19].In commercial salmonid production, for example, several transport methods can be utilized, and the most common are coastal transport by means of well-boats or towing of culture cages, road transport of isolated tanks, and transport by helicopter over short distances [20].
Fish can be transported in boxes, bags, or improvised boxes, yet it is still possible to find transport being performed inappropriately in improvised containers without any professionalism.The material of the transport containers can influence the maintenance of the water temperature, and many are made of fiberglass or aluminum, containing an insulating material such as pressed polyurethane [21].Additionally, using fiberglass tanks is thought to be non-toxic to fish, simpler to manage, and easy to clean [22].Ornamental fish with high commercial value that are meant for export are packed in polyethylene bags that are saturated with pure oxygen.The transport water is usually pre-treated with chemicals or medications.The bags are then sealed and put inside styrofoam (polystyrene) boxes to act as thermal insulation [23].
The success of transport is directly related to the quality of the animals being transported.This, in turn, is associated with the conditions in which the animals are produced prior to the procedure, which implies the use of good production practices, cultivation conditions, water quality, feeding, and care during harvest.For this stage to be successful, the species and its requirements in relation to the quality of the transport water must be known.The fish size can also have direct implications for management, as well as the need for fasting, the densities that can be used, the type of transport, and the fish's tolerance to the use of products such as salt and anesthetics, among other management approaches, most of which will be covered in this review.The need for a holistic view, with broad knowledge about physiology, water quality, and the procedures to be adopted for efficient transport with minimal animal stress and loss, is evident.This review will address the main aspects of the transport of live fish using salt and anesthetics and different stocking densities.

Water Quality during Fish Transport
During transportation, fish may be subjected to a variety of stressful situations, such as poor water quality, which can seriously impair animal physiology and survival [24][25][26][27].Transport-related considerations for critical water quality indicators include temperature, ammonia, carbon dioxide (CO 2 ), pH, and dissolved oxygen (DO).
The primary organ of aquatic respiration for the majority of fish is their gills, which are made to efficiently transport carbon dioxide out of the blood and oxygen from the water into the blood [28].Since fish require more oxygen during long transports, especially when stocking densities are high and the duration of the transport is extended, dissolved oxygen is thought to be the most restricting element during fish transport and must always be maintained at an appropriate level throughout the process [21].In order to accomplish this, animals can be carried in a variety of containers that have aeration or compressed air injection [29] or in plastic bags that have 1/3 of the capacity filled with water and the air replenished with pure oxygen [30].
Carbon dioxide (CO 2 ) is a byproduct of cellular oxidative metabolism and is highly soluble in blood and water, being excreted mainly through the gills [21,31].However, high concentrations of CO 2 in transport water can be very harmful to fish, resulting in hypercapnia-that is, high levels of CO 2 in the blood-followed by respiratory acidosis and a reduced oxygen-carrying capacity (Root effect) and affinity for hemoglobin by oxygen (Bohr effect), thus promoting tissue hypoxia and animal death [21,[32][33][34][35].One of the main effects of plasma acidosis is impairment of the blood's ability to transport oxygen [36].
The gills perform important functions in addition to gas exchange, such as osmoregulatory functions, acid-base balance, and the excretion of nitrogenous compounds [37], with ammonia as the main product of protein catabolism and metabolism.Ammonia occurs in two chemical forms in water: ionized (NH 4 + ) and non-ionized (NH 3 ), the latter being considered the most toxic form, as it easily diffuses through the gill epithelium of fish.Fur-thermore, ammonia toxicity is significantly affected by pH, as an increase in pH induces an increase in the concentration of ammonia in the NH 3 form [38,39].When at high levels, ammonia can promote reduced growth, oxidative stress, immunosuppression [40][41][42][43], and fish mortality [44].However, the use of some products in transport water can mitigate increased ammonia.Zeolites, for example, are microporous crystalline hydrated aluminosilicates that can serve an important role in removing total ammonia nitrogen in transport tanks [45].The use of zeolites showed an increase in the survival rate for species such as Catla catla (Cypriniformes:Cyprinidae), Labeo rohita (Cypriniformes:Cyprinidae), Cirrhinus mrigala (Cypriniformes:Cyprinidae) [46], and Ancistrus triradiatus (Siluriformes:Loricariidae) [47] and allowed stocking densities to be increased and improve the well-being of juvenile Dicentrarchus labrax (Eupercaria:Moronidae) [48].The use of 15 g of zeolite and 5 g of activated carbon in water also increased the survival rate of juvenile Chanos chanos (Gonorynchiformes:Chanidae) during a 36 h transport [49].
Pre-transport fasting has also been widely used and shown to be highly effective in reducing ammonia excretion by fish [50][51][52][53], as fasting reduces the rate of metabolism and thus reduces oxygen consumption and waste production by these animals [52].Different fasting periods are recommended for transporting fish; for example, a period of 36 h was considered ideal for transporting Gadus morhua (Gadiformes:Gadidae) at a water temperature of 12 • C [53], while juveniles of Cyprinus carpio (Cypriniformes:Cyprinidae), fasted for up to six days before transport, were able to regulate the transport of ammonia and ions efficiently and their energy reserves (liver glycogen) were not compromised.However, the authors state that factors that affect metabolism, such as the temperature and fish size, should be considered in future studies [52].
The water's pH is also an important factor in need of attention when transporting fish, as variations in CO 2 and ammonia concentrations are directly related to the pH [29].Fish respiration encourages the buildup of CO 2 , which causes the water to become more acidic as transport times grow.As was previously indicated, the acidic pH also causes a decrease in the fish's blood's ability to carry oxygen, even though this acidification reduces the amount of ammonia in the water in its harmful form (NH 3 ).These differences can be lessened, though, by using buffers in the water, such as sodium bicarbonate, tris buffer [tris-(hydroxymethylaminomethane)], and magnasphere diffusers or by maintaining a pure oxygen environment in transport containers [33,54].The use of 2.5 g/L of sodium bicarbonate, 1 g/L of TRIS, and 13 g/L of magnaspheres showed efficient buffering in the transport water of G. morhua, with an average weight of 28.6 ± 2.5 g [54].For the species Scomber japonicus (Scombriformes:Scombridae) and Sarda sarda (Scombriformes:Scombridae), transported for 25 h both by air and road, it is recommended to add 40 ppm of bicarbonate and sodium carbonate every 3 h of transport for efficient water buffering [13].
Changes in water temperature during transport are also expected, which directly influence respiratory responses and O 2 consumption by fish, as they are ectothermic animals [55,56].Temperature control refers to keeping the water's temperature within a predetermined range while it is being transported.As previously indicated, insulated boxes, temperature-controlled box trucks, refrigerators, and/or ice can all aid in this process.A rise in the metabolic rate brought on by water temperatures over the range that is optimal for a particular species of fish might result in thermal stress circumstances, where fish need the energy to cope with stress responses and so have a lower probability of surviving [57].
Strategies, such as reducing the water temperature during transport, have been used with the aim of reducing the metabolic rate and oxygen consumption and increasing the solubility of oxygen in water, in addition to providing a decrease in the production of nitrogenous waste by the animals [56,58,59].However, there is a tolerable limit of reduction in transport water temperature for tropical fish, which is 22 • C and 15 to 18 • C for temperate species [23].Another important strategy to be considered to avoid sudden fluctuations in water temperature is the correct packaging of the animals during transport-for example, the use of styrofoam (polystyrene) boxes to place the plastic bags in which the fish will be packed, allowing efficient thermal insulation and, consequently, smaller variations in water temperature [23].
Different temperatures (15, 20, and 25 • C) and storage densities (50, 67, 87, and 168 g/L) were evaluated in the 24 h transport of Rhamdia quelen (Siluriformes:Heptapteridae) fingerlings (1.0 to 2.5 g) in plastic bags [56].This study found that even at the maximum stocking density of 168 g/L, fish survival was increased by the cooler water (15 • C).The authors further claim that because of the low oxygen content and high carbon dioxide concentrations in the water, transport cannot last longer than six hours at a temperature of 25 • C. The use of a low water temperature (15 ± 1 • C) for the transport of Coreius guichenoti (Cypriniformes:Gobionidae) (165.84 ± 38.62 g) promoted mitigation of the effects of stress after transport by reducing cortisol levels and preventing an increase in levels of blood lactate when compared to the ambient temperature of 22 ± 1 • C [58].

The Use of Salt during Fish Transport
Salt (NaCl) has been a product widely used in fish production for various purposes [59,60], including transport.Salt is cheap, easy to acquire, and non-toxic for the handler, in addition to being permitted for use in aquaculture.In freshwater fish, salt can improve the electrolyte balance (sodium Na + and chloride Cl -ions) [61], reducing the gradient between water and blood, mitigating stress responses, promoting well-being, and preventing diseases [7,47].This review addresses the use of salt during transport and its impact on physiological responses in different species of fish, as well as possible changes in water quality.
The use of 8 g of salt/L in water for the transport of Colossoma macropomum (Characiformes:Serrasalmidae), weighing 846.0 ± 25.0 g, in cylindrical plastic boxes for 3 h at a density of 65 kg/m 3 , was efficient at decreasing most physiological stress responses when compared to using 0, 2, and 5 g of salt/L.With 8 g of salt/L, there was no change in cortisol and glucose concentrations, while cortisol values for treatments with 0, 2, and 5 g of salt/L were higher at 24 h after transport, returning to basal levels only 96 h after transport.There was also an increase in plasma glucose in treatments with 0, 2, and 5 g of salt/L after transport, returning to baseline values 24 h after transport.However, despite the stress, the fish in all treatments satisfactorily accepted food at around 18 h after transport and normally fed after 48 h [6].However, the use of salt at concentrations of 1, 2, and 3 g/L for juveniles of the same species, weighing 1.19 ± 0.06 g, transported in plastic bags at densities of 15 to 180 fish/L and for periods of 3 to 24 h, did not improve post-transport survival [62].The results suggest different responses depending on the fish size, the transport method, or the duration used.
Salt was also efficient in the transport of the gold-spot (A.triradiatus) weighing 10.4 ± 4.6 g.The fish were transported in plastic bags, with the inclusion of salt at concentrations of 0.5 and 1 g/L at a fish biomass of 61.75 g/L.The use of salt promoted a reduction in ammonia levels in the water, did not change blood glucose concentrations after transport, and presented lower mortality up to the seventh day post-transport.Based on these and other results, the authors recommended the use of 1 g/L of salt in the transport water for the species [47].
The transport of juvenile Labeo victorianus (Cypriniformes:Cyprinidae) for 6 h in plastic bags with different salt concentrations (0, 0.25, 0.5, 1, 2, 4, 8, and 10 psu) at a biomass of 16 kg/m 3 found higher dissolved oxygen levels for salinities of 1 to 4 psu, with the highest level being at 10 psu of salt.The concentrations of total ammoniacal nitrogen and CO 2 were lower for the higher salinities.Transport with salt concentrations between 1 and 4 psu experienced no mortality.Cortisol was highest with 0 and 0.25 psu, while between 0.5 and 8 psu, there were no changes.Glucose levels were highest between 0 and 0.5 psu and for 8 and 10 psu.Sodium and chloride levels and plasma osmolarity were altered for concentrations of 0, 0.25, 0.5, and 10 psu.Thus, there is a correlation between water parameters and physiological responses, with the use of 0.5 to 8 psu of salt being able to mitigate cortisol production, while levels of 1 to 4 psu of salt were more efficient at maintaining other blood parameters [63].
Additionally, juvenile L. rohita weighing 4.6 ± 0.5 g and carried for 12 h at a density of 100 g/L showed that salt was effective at mitigating the release of cortisol.Moreover, the serum levels of Na + and Cl − ions were lowest in treatments of 0 and 2 g NaCl/L.After being transported with 4 g of NaCl/L, the enzyme glucose-6-phosphatase showed minimal activity on the first and second sampling days.The activity of the enzymes lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) in muscle tissue was high in the control group, while the 4 g concentration exhibited the lowest values, in addition to greater survival, being the best-recommended concentration with the aim of mitigating stress and mortality in these animals [61].
Juvenile lambari (Astyanax altiparanae) (Characiformes:Characidae) were transported in concentrations of 0, 3, 6, and 9 g of salt/L for 8 h in plastic bags (45 × 27 cm) at densities of 22, 30, and 37 g/L.The estimated salinity of 8.14 g/L led to the lowest water pH.Plasma glucose was also affected, with higher levels in fish kept in fresh water and at the highest density (37 g/L) and the lowest levels being found at estimated salinities of 5.96 and 6.40 g/L and for densities of 30 and 37 g/L, showing that the addition of salt to water also reduces stress responses in this species [64].Elevated glucose levels were also found in triploid female rainbow trout (average weight 200 g), transported for 5 h in fresh water, compared to a concentration of 5 g NaCl/L.In addition, the use of salt promoted a reduction in the excessive growth of bacteria associated with the skin.Thus, the authors reported that salt could have a beneficial effect on the microbial communities of fish and highlighted the importance of mucosal health during handling, such as transport [65].
Salt was also effective for killifish (Hypsolebias flagellatus) (Cyprinodontiformes:Rivulidae) weighing 1.38 ± 0.83 g transported individually in plastic bags with 0, 2, 4 and 6 g salt/L for 12 h.No mortality was observed up to 96 h after transport, although the physiological parameters of the fish were not evaluated.The water quality was similar between treatments, with the exception of dissolved oxygen, which was found to be higher when using 4 g of salt/L and lower when using 0 g of salt/L [66].
Despite the benefits cited in previous studies, salt was not efficient in mitigating the effects of stress in the transport of arapaima (Arapaima gigas) (Osteoglossiformes: Arapaimidae) weighing 32.79 ± 2.35 g, transported in plastic bags at a density of 40 g/L and in salinities of 1, 3, and 5 g/L for 3 h.Although no mortality was observed, there was an increase in cortisol after transport with a concentration of 1 g of salt/L, and within 24 h after transport, this increase occurred for all treatments, returning to basal levels 48 h after transport.Glucose levels also increased after transport, returning to basal levels 24 h after transport.The authors also suggest that there is a long latency period in the cortisol response for this species [67].This latency was also verified for animals of this species weighing 1 kg, transported in an open system in styrofoam boxes or individually enclosed in plastic bags, using 0, 3, and 6 g of table salt/L of water for 3 h.There was no mortality 48 h after transport.However, in both systems, cortisol was higher for salt use at 24 and 48 h after transport [7].The use of 3 g of salt/L showed a better response for this species with 752.0 ± 48.0 g, transported in polyethylene bags at a density of 375 g/L, for 5 h.Osmolarity, cortisol, glucose, and total proteins were similar among treatments.However, Na + and Cl − values were higher for 3 g of salt/L, indicating a better response to transport stress through lower energy expenditure to maintain osmoregulation [68].
Pacamã (Lophiosilurus alexandri) (Siluriformes:Pseudopimelodidae), transported with 0, 2, 4, 6, and 8 g of salt/L at a density of 10 fish per plastic bag for 5.5 h, did not show mortality up to 48 h after transport.Furthermore, dissolved oxygen was higher in 0 and 2 g of salt/L, while ammonia concentrations were lower in water without added salt (0 g/L).After 24 h of transport, concentrations of 6 and 8 g of salt/L showed a reduction in glucose levels and, after 48 h, all treatments were similar to baseline.Hematocrit was lower after transport at concentrations of 6 and 8 g of salt/L.As there was also no better physiological condition of the animals with 2 and 4 g of salt/L compared to the control, this additive was not efficient in mitigating stress for this species [69].
For juvenile panga (Pangasianodon hypophthalmus) (Siluriformes:Pangasiidae) weighing 22.96 ± 5.38 g transported for 5 h in plastic bags containing a density of 1 juvenile/L, salinities of 4 and 8 g/L did not show the potential to reduce stress.Even without mortality up to 96 h after transport, the use of 4 g of salt/L increased cholesterol after transport, returning to basal levels within 24 h post-transport, while glucose was higher for 4 g of salt/L after transport and for 8 g of salt/L at 24 h after transport.Triglyceride levels were elevated after transport with 4 g of salt/L, remaining unchanged for up to 96 h [70].
Another catfish, jundiá (Rhamdia quelen) (Siluriformes:Heptapteridae), weighing 360.0 ± 99.0 g, was tested in salinities of 8 and 12 g of salt/L at a density of 150 kg/m 3 for 3 h of transport and showed no difference in cortisol between treatments.High levels of Na + and Ca 2+ were recorded in both salinities, compared to the use of fresh water, while 12 g of salt/L provided a lower concentration of chloride in plasma.Furthermore, 12 g of salt/L contributed to an earlier onset of total rigor mortis, having a negative impact on the quality of the fish meat [71].When the same species, measuring 5.8 ± 0.8 cm, was transported in polyethylene bags at a density of 100 g/L in salinities of 0, 1, 3, and 6 g/L for periods of 6, 12, and 24 h, low levels of dissolved oxygen were recorded (1.0-2.5 mg/L) for the 24 h transport, and there was total mortality in treatments using salt.However, there was no mortality during transport up to 6 h.After 12 h, mortality increased with an increasing salt concentration.Changes in body Na + and K + levels were recorded in salt treatments, confirming that it is not a good additive for this species [72].The use of salt was also not efficient in transporting the cardinal tetra (Paracheirodon axelrodi) (Characiformes:Characidae) in plastic boxes at a density of 3.23 ± 0.4 g of fish/L.The use of tetracycline + table salt (2.5 mg/L + 66.67 mg/L, respectively) for 24 h provided high survival (99.5%); however, it led to a very high influx of Na + , as well as increased Cl − efflux and increased K + influx, compromising the ionoregulation of the cardinal tetra [15].
Table 1 presents the recommended concentrations of this additive for different fish species.Thus, given the studies mentioned above, the use of salt in transport water is highly recommended for many species of fish, as it directly mitigates stress responses, such as decreasing cortisol levels.It also contributes to improving iono-osmoregulatory balance and reduces mortality, in addition to improving the quality of transport water.However, it is necessary to consider that the best salt concentration to be used for a given species depends on factors such as animal size, as well as the method, type, and duration of transport.Furthermore, it is noted that there are phylogenetic differences in relation to the use of salt for transport.Fish from the order Siluriformes are less tolerant to high concentrations of salt in the transport water when compared to Characiformes and Cypriniformes, for example.Regarding the studies mentioned in Table 1, it is also observed that freshwater fish, except for Siluriformes, can tolerate concentrations of up to 8 g/L of salt in transport water, while diadromous fish can tolerate concentrations of up to 10 g salt/L.

The Use of Anesthetics during Fish Transport
Anesthetic agents have been used in aquaculture with the aim of reducing the impacts of stress during transport and handling, such as weighing, classification, vaccination, blood collection, and experimental surgeries [81][82][83].This can lead to improved animal health and well-being and reduced morbidity and mortality, as well as greater economic yield in production [84].When used without caution, anesthetics can lead to negative consequences for animals, such as acidosis, osmotic stress, insufficient exchange of gases and ions between blood and water, and respiratory arrest [82].
For their safe use in aquaculture, anesthetics must be easy to administer, the animals' recovery must be rapid, and they must not produce lasting physiological effects, with rapid removal from the organism.Furthermore, they must have high solubility in fresh and salt water, be readily available and economical, and not generate toxicity for humans.A wide variety of anesthetics have been used in several studies on fish transport, including synthetic anesthetics, such as tricaine methanesulfonate (MS-222), benzocaine, lidocaine, metomidate, and propofol, and natural anesthetics, such as plant essential oils and extracts (Table 2).
Tricaine methanesulfonate, known as MS-222, is a crystalline, white, water-soluble powder derived from benzocaine [86].It is considered an anesthetic capable of blocking nociceptive pathways in procedures that may cause pain [82], promoting rapid anesthesia and animal recovery [131], despite being considered expensive and requiring special preparation and storage [132].The addition of 40 mg/L of MS-222 improved water quality due to decreased ammonia nitrogen and promoted a decrease in serum cortisol for yellow catfish (Pelteobagrus fulvidraco) (Siluriformes:Bagridae) transported for 12 h [88].It also reduced stress in the juvenile of Oreochromis niloticus (Cichliformes:Cichlidae) transported for 2 h [8] and in the ornamental Indian tiger barb (Puntius filamentosus) (Cypriniformes:Cyprinidae) during transport for 48 h [87].In addition to decreasing stress, a concentration of 30 mg/L of MS-222 in water during the transport of sea bass (Lateolabrax maculatus) (Acropomatiformes:Lateolabracidae) resulted in better meat flavor and quality [9].However, simulated transport of crucian carp (Carassius auratus) (Cypriniformes:Cyprinidae) for 2, 4, 8, 12, and 24 h, exposed to concentrations between 30 and 45 mg/L of MS-222 in water, positively impacted the animals' immune system due to a greater probability of inducing the expression of immune genes [89].In another study with MS-222, juvenile rabbit fish (Siganus rivulatus) (Acanthuriformes:Siganidae) subjected to 24 h transport showed abrupt movements during periods of exposure and recovery, in addition to mortality [85].
Benzocaine is an anesthetic widely used in human and veterinary medicine as a local analgesic.It is administered to fish for sedation, immobilization, analgesia, and general anesthesia or narcosis and presents the same properties of blocking nociceptive pathways as MS-222 and isoeugenol [82].The use of 20 mg/L of benzocaine in water improved post-transport survival and consequently reduced economic losses in the production of the black-spot barb (P.filamentosus) [87].In a study involving the transport of juvenile panga (P.hypophthalmus) for 5 h, the use of 5 mg/L of benzocaine was sufficient to exert a sedative effect on the fish, in addition to significantly reducing the excretion of ammonia into the water by the animals [70].The combination of 12 mg/L of benzocaine and water hypothermia promoted a reduction in the loss of scales during the transport of the Mexican silverside (Menidia estor) (Atherinopsidae) for 3.5 h [93].
While many studies demonstrate that benzocaine reduces stress during transportation, benzocaine at concentrations of 5, 10, and 20 mg/L did not prevent an increase in plasma cortisol levels and adversely affected hematological variables and the number of gill parasites of matrinxã (Brycon cephalus) (Characiformes:Bryconidae) [133].This study, which involved juvenile cobia (Rachycentron canadum) (Carangiformes:Rachycentridae) transported in water containing 2 or 6 mg/L of benzocaine, is also supported by the findings of [134].Metomidate hydrochloride (metomidate-HCl) is an imidazole-based compound that is effective for animal anesthesia but not for analgesia [94].The use of 1 mg/L of metomidate-HCl in transport water for the Congo ornamental cichlid angelfish (Cichlasoma nigrofasciatum) (Cichliformes:Cichlidae) showed promising responses in reducing mortality [135].This same concentration of metomidate also reduced stress in Atlantic salmon (Salmo salar) (Salmoniformes:Salmonidae) during a 2 h transport [95].Koi carp (Cyprinus carpio) (Cypriniformes:Cyprinidae), transported for 24 h in water containing 3.0 mg/L of metomidate, experienced an inhibitory response to plasma cortisol, with no adverse effects on animal behavior [94].
Lidocaine hydrochloride (lidocaine-HCl) is a water-soluble anesthetic widely used in the dental industry [96]; however, there are few investigations into its effectiveness in fish transport.The most recent study found that this compound reduced metabolic activity in flounder (Pleuronectes americanus) (Pleuronectiformes:Pleuronectidae), which, consequently, reduced the excretion of ammonia in the water and lowered oxygen consumption [96].
Regarding the low cost and accessibility of synthetic anesthetics, propofol (2,6diisopropylphenol) has been recommended as a priority in the transport of ornamental fish, as was the case for the species jack dempsey (Rocio octofasciata) (Cichliformes:Cichlidae), in which it proved to be more effective than a natural anesthetic based on clove oil [136].Propofol has sedative and anesthetic effects, with ultra-short action but without analgesic properties.It has also been shown to be beneficial in reducing stress at a concentration of 0.8 mg/L without compromising the health, well-being, or quality of Nile tilapia fillets during a 6 h transport [97].The concentration of 0.4 mg/L of propofol in the transport water for juvenile catfish (R. quelen) was able to maintain ionic and respiratory homeostasis and prevent peroxidative damage to vital organs [98].
In addition to synthetic anesthetics, a wide variety of plant essential oils have been used as potential natural anesthetics in aquaculture.However, the largest number of studies corresponds to the essential oil obtained from the distillation of the leaves, flowers, stems, and buds of clove (Eugenia aromatica, Eugenia caryophyllata or Syzygium aromaticum) [137], its main component being eugenol (4-allyl-2-methoxy-phenol).The transport of juvenile panga (P.hypophthalmus) [70] and Korean bullhead (P.fulvidraco) [84] with the use of 10 mg/L of eugenol provided a decrease in ammonia excretion and reduced fish stress responses, respectively.Angelfish (Pterophyllum scalare) (Cichliformes:Cichlidae), transported for 7 h in water containing a concentration of 15.9 mg/L of eugenol, experienced a reduction in ammonia excretion, and the fish did not show histopathological changes in the gills [101].
In a study aimed at the use of essential oils during pre-slaughter transport of O. niloticus, the use of 20 µL/L of eugenol delayed the drop in muscle pH and did not influence the texture, luminosity, and profile of fatty acids of the fillets of these animals [99].Furthermore, less damage to the liver of juvenile sea bass (L.maculatus) was observed during transport with 6 mg/L of eugenol when compared to the use of the synthetic anesthetic MS-222 [103].However, species such as carp (C.carpio) did not show satisfactory responses to the use of 5 mg/L of clove oil during long-term transport (24 h), as oxidative stress was observed in the gills and liver of these animals, in addition to greater excretion of ammonia in the water [106].
The plant named Lippia alba, popularly known as bushy matgrass, is found from southern North America to southern South America and Africa [114,138] and is considered safe for consumers and the environment [112].Many studies show the benefits that its essential oil presents during fish transport, such as improving oxidative stress and the well-being of juvenile R. quelen [10,104], reduced stress in juvenile Nile tilapia [112] and juvenile tambacu hybrids (Piaractus mesopotamicus × C. macropomum) (Characiformes:Serrasalmidae) [114], in addition to reducing ammonia excretion in water and improving the quality of tambacu fillets [114].
The essential oil of Ocimum basilicum, from basil, which is distributed in South America and Asia [139], has also proven effective when used during fish transport.It reduced total ammonia nitrogen in the water, energy metabolism, and damage to the liver of groupers (E.fuscoguttatus × E. lanceolatus) (Perciformes/Serranoidei:Epinephelidae) [117] and delayed the drop in muscle pH, without affecting texture and profile of fatty acids in Nile tilapia fillets [99].Furthermore, juvenile tilapia transported for 2 h showed a lower residual concentration of O. basilicum in muscle tissue when compared to eugenol [100].The essential oil of Ocimum gratissimum, from the alfavaca plant, has been studied for inclusion in the transport water of juvenile C. [118], O. niloticus [119], L. alexandri [70], and Paralichthys orbignyanus (Pleuronectiformes:Paralichthyidae) [121].The concentration of 5 mg/L was recommended for the transport of species O. niloticus and C. macropomum, and the concentration of 10 mg/L for O. gratissimum was indicated as the best concentration for C. macropomum, L. alexandri, and P. orbignyanus.
The plant Aloysia triphylla, known as lemon verbena, promoted a reduction in total levels of ammoniacal nitrogen, maintained dissolved oxygen at adequate values, and reduced the ventilatory frequency of fish, in addition to improving the physiological parameters of juvenile catfish L. alexandri during transport [140].Similar responses regarding improved physiological conditions were also found for the silver catfish (R. quelen) [123].The use of 30 µL/L of essential oil of A. triphylla in water reduced the loss of ions (Na + , Cl − , and K + ) by juvenile Nile tilapia during transport for 8 h [122].
Based on the studies previously presented on the use of anesthetics in fish transport, it is observed that there is a huge variety of synthetic and natural anesthetics that have great potential to improve the well-being of fish by mitigating stress responses, reducing mortality during and after transport, in addition to promoting great benefits in the quality of transport water.However, it is necessary to take into account that the choice for the best anesthetic must be made based on its availability, accessibility, and cost, whether synthetic or natural, in addition to knowing the best concentration to be used and recommended for a given anesthetic for the species to be transported.

Stocking Density and Fish Transport
Stocking density is another important factor to be considered during fish transport since an inadequate density can stress fish, worsen water quality, and lead to fish death.Fish subjected to high stocking densities during transport may present bodily injuries, such as skin lesions after the land transport of Clarias gariepinus (Siluriformes:Clariidae) [141] and corneal opacity in Micropterus salmoides (Centrarchiformes:Centrarchidae) [142], related to contact with the eyes of these animals with spines and scales from other animals crowded in the same transport tank [143].
The table below (Table 3) presents the recommended stocking density for the transport of freshwater and marine species.
The transport of jundiá (R. quelen) has been carried out with fish of different weights and sizes.Fish weighing 2.55 ± 0.44 g were transported for 4, 8, and 12 h, at densities of 30 and 60 fish/5 L and at temperatures of 15, 20, and 25 • C. The temperature and time had significant influences on ammonia, pH, electrical conductivity, and oxygen levels, and mortality was observed after 12 h of transport simulation at 20 and 25 • C. The density directly influenced water-quality parameters, such as increasing electrical conductivity and turbidity and decreasing dissolved oxygen and pH at a density of 60 fish/5 L. Therefore, transport is recommended at temperatures between 15 and 25 • C for periods of less than 12 h and at a density of 6 fish/L of water [153].Jundiá (R. quelen) measuring 5 to 10 cm were transported in plastic bags at 15, 20, and 25 • C, in densities of 50, 67, 87, and 168 g/L and for durations of 6, 12, and 24 h.Mortality was significant for the density of 168 g/L after 24 h of transport at 20 and 25 • C. Dissolved oxygen was also reduced in these treatments.The temperature of 15 • C was the best, while transport could not exceed 6 h at 25 • C and at the highest density.Survival was 100% for 24 h with densities of 50, 67, and 87 g/L at all temperatures [56].The results indicate the great importance of the density and water temperature for the transport of this species.However, for fish weighing 23.2 ± 5.3 g, transported in plastic bags for 4 h, at densities of 75, 150, 250, and 350 g/L, ammonia increased with increasing densities, and dissolved oxygen was lower for the highest density tested.Cortisol was also higher after transport at the highest density; however, it returned to baseline levels after 24 h.Glycemic levels were also influenced by density, but without mortality, and the use of 350 g of salt/L is recommended for this transport time (4 h) [60].Changes in water quality were also recorded when jundiá (76.6 ± 0.7 g) were transported for 5 h in plastic bags, in which densities of 221, 286, and 365 g/L were tested.Dissolved oxygen was lower at the highest density, while non-ionized ammonia was higher at the two highest densities, with impacts on the fish, as a positive relationship was found between the flow of ions and density and a negative relationship between ammonia excretion and density [154].
Weighing 1000.0 ± 250.0 g, adult matrinxã (B.cephalus) were moved for 4.5 h in plastic containers filled with water containing 6 g NaCl/L at densities of 100, 200, and 300 kg/m 3 .As long as 96 h after transit, no deaths were noted.However, because of a rise in glucose and a fall in serum sodium and plasma chloride levels, the animals were stressed by the 300 kg/m 3 density.High densities can, therefore, be employed, although more caution is required to prevent the animals from suffering further stress [149].Nevertheless, after being transported for four hours and being kept in plastic bags with densities of 83, 125, 168, and 206 g/L, young matrinxã (23.5 ± 0.4 g) did not exhibit any mortality following the travel or following a week of monitoring.Although there was an increase in cortisol and glucose levels for all densities after transport, both returned to baseline levels within 24 h after transport, allowing the highest density tested to be recommended for use [147].
In a similar study with the same species (B.cephalus) but of lower body weight (13.3 ± 4.9 g), the animals were stored in polyethylene bags at densities of 83, 125, and 166 g/L for 4 h.As in previous studies, there was no mortality.Glucose and cortisol also increased after transport in all treatments but also returned to baseline levels after 24 h.The authors also recommended using the highest density tested for this species [148].The results showed that, despite the stress of transport, the fish returned to homeostasis within 24 h after transport, returning to the intake of feed, and that the stress produced did not seem to be so high that higher densities could not be used.
Tambaqui (Colossoma macropomum) (Characiformes:Serrasalmidae), weighing 846.0 ± 25.0 g, were transported in cylindrical plastic boxes with supplementary aeration to maintain dissolved oxygen between 4.0 and 8.0 mg/L, for 3 h, at densities of 100, 150, and 200 kg/m 3 , with 8 g of salt/L also added to the water.There was no change in plasma cortisol levels for all densities tested, which was attributed to the use of salt.The fish in the two lowest densities accepted food at around 18 h after transport, and in the highest density, there was 11% mortality in one of the replicates, in addition to the presence of injuries, slow swimming, and lower food consumption in some fish during the first 48 h after transport, such that a density of 200 kg/m 3 is not recommended [6].High densities are not recommended for the transport of juveniles of this species with lower weight (51.9 ± 3.3 g) in polyethylene bags at densities of 78, 156, 234, and 312 kg/m 3 for 10 h.Cortisol and glucose showed higher values in the three highest densities after transport, with accumulated mortality of 32, 43, and 65% for the three highest densities, respectively, after 96 h [146].However, for fish weighing 1.12 ± 0.28 g, transported at densities of 30 (33.60 ± 9.40 g/L), 60 (67.20 ± 16.8 g/L), and 90 (100.8 ± 25.2 g/L) fish/L for 17 h, the use of essential oil of Lippia alba (20 µL/L) was effective for the highest densities (60 and 90 fish/L), reducing conductivity and with a lower loss of ions by fish compared to the use of water without the essential oil [145].
These results indicate that the density to be used for this species is related to the size of the animals and that the use of additives can be more efficient for smaller fish, enabling the use of higher densities.
Among Neotropical catfishes, pacamã (L.alexandri), weighing 2.1 ± 0.6 g, were transported for 11 h using water with a salinity of 1.6 g/L, at densities of 40, 55, and 70 juveniles per bag, containing 8 L of water.The higher density led to mortality of the animals at 24 and 48 h post-transport [157].A study with another catfish, the yellow mandi (Pimelodus maculatus) (Siluriformes:Pimelodidae), weighing 5.72 ± 1.55 g, stocked at densities of 4 (22.88 g/L), 8 (45.76 g/L) and 12 (68.64g/L) fish/L, transported for 4, 8, and 12 h, found no changes in dissolved oxygen levels, while ammonia was higher at the highest density.However, the absence of differences in cortisol levels indicates that the transport conditions were appropriate [158].Thus, variation among studies using the same species, but with differences between animal size, transport time, and densities, or even between different species, indicate the need for individual assessments.
Juvenile pirarucu (A.gigas), weighing 488.0 ± 26.5 g, transported for 4 h in plastic boxes at densities of 98,146,195, and 244 g/L, did not show mortality within 48 h after transport.Hematocrit increased with densities of 195 and 244 g/L, with subsequent recovery.The highest levels of cortisol after transport were found in fish at densities of 98 and 146 g/L.At 24 h after transport, there was a drop in cortisol levels, with greater cortisol latency for densities of 195 and 244 g/L.Nonetheless, the authors recommend using the highest density for this species [144].A similar finding was reported for juvenile A. altiparanae transported for 8 h at densities of 22, 30, and 37 g/L in plastic bags and with the addition of salt to the water of up to 9 g/L.Transport in fresh water and at higher density increased plasma glucose levels.Despite increased density promoting reduced dissolved oxygen levels in the water, there was no mortality of the animals [64].
A density of 81 kg/m 3 can be recommended, over densities of 27 and 54 kg/m 3 for the safe transport of marine fish golden pompano (Trachinotus ovatus) (Carangiformes:Carangidae) weighing 3.38 ± 0.36 g in polyethylene bags, for 8 h and with the addition of 7 mg/L of eugenol, as physiological and biochemical parameters were not affected [160].For another marine fish, known as orange-spotted grouper (Epinephelus coioides) (Perciformes/Serranoidei: Epinephelidae), weighing 3.0 ± 0.2 g and transported at densities of 20, 30, 40, and 50 no/L for 6 h, several biochemical (glucose, lactate dehydrogenase, aspartate amino transferase, and alanine amino transferase) and water quality changes were recorded, but with values within the tolerable range and without mortality, with the highest density tested being recommended by the authors [162].A higher transport density also seemed to have a positive effect on O. niloticus weighing 866.86 ± 143.98 g.The fish were transported at densities of 100 and 400 kg/m 3 for 3 h with the addition of 6 g of salt/L.Cortisol was similar to the basal group after transport, and there was no effect on fillet quality, with 400 kg/m 3 showing better visual acceptance by the panelists [156].
Higher densities might not be recommended for juvenile dourado (Salminus brasiliensis) (Characiformes:Bryconidae) weighing 0.71 ± 0.53 g and stored at densities of 5, 10, and 15 g/L during transport of 4, 8, and 12 h.Fish experienced the highest ammonia in water for a density of 10 g/L, while the concentration of nitrite was highest at 15 g/L.After 4 h of transport, cortisol was higher in all densities compared to the baseline.However, during longer transport times, cortisol concentrations decreased for the lowest density and remained higher at 10 and 15 g/L.Even if there is no mortality, higher densities or longer transport times can be harmful to the species [150].
Cortisol took longer to return to the basal level after transporting flounder (Pseudopleuronectes americanus) (Pleuronectiformes:Pleuronectidae) with a total length of 42 mm at the highest density tested (600%, corresponding to the surface area of the substrate and estimated as a function of the flat shape of the flounder), compared to densities of 100, 200, 300, and 400%.Thus, the study recommends using a density of up to 400% with an acclimation period of at least 48 h for these animals [163].Physiological changes and mortality were also reported in juvenile rohu (L.rohita) measuring 14 to 15 cm in length, transported in polyethylene bags for 2 h and 30 min, at a density of 201 g/L, when compared to 67 and 134 g/L.Furthermore, the ammonia concentration was also higher at the highest density [151].
Silver carp (Hypophthalmichthys molitrix) (Cypriniformes:Xenocyprididae) weighing 5 g were transported with pure oxygen and atmospheric air at four densities (30,60,90, and 120 g/L) in three different forms of containers (polyethylene bags, cuboidal, and cylindrical) and for three transport durations (2, 4 and 6 h).Water oxygen levels varied from 0.67 to 7.11 mg/L for the four densities tested.Ammonia ranged from 1.1 to 7.0 mg/L, with the lowest value being recorded for the density of 30 g/L and 2 h of transport, with pure oxygen supply and in polyethylene bags.The lowest survival occurred for the highest density with an atmospheric air supply and a 6 h transport duration.Therefore, the authors recommend transportation for 2 h in polyethylene bags at a density of 30 g/L with a supply of pure oxygen [152].Higher densities, associated with higher temperatures, were also not efficient for transporting grouper larvae (Epinephelus sp.) (Perciformes/Serranoidei:Epinephelidae).The density of 50 larvae/L was best for larvae aged 45 and 60 days for 8 h transport at 23 • C.However, up to 100 larvae/L can be used during 8 h of transport at 23 • C, with mortality varying between 2.3 and 5.3%.The temperature of 28 • C and densities of 100 to 200 larvae/L resulted in increased NH 3 levels and mortality.Therefore, the authors recommend transport at a density of 50 grouper larvae/L, aged 60 days, at a temperature of 23 • C [4].
The review of these studies makes it clear that there are numerous factors that can influence the best stocking density for transport.The most significant among these include the transport duration, as many studies show that long-term transport under high densities tends to promote greater stress for animals and worsen the water quality.The water temperature is another crucial factor during fish transport, as temperatures above those considered ideal for a given species, combined with higher stocking densities, can drastically reduce dissolved oxygen concentrations and increase toxic ammonia levels in the water, consequently promoting stress and animal mortality.However, for some fish species covered in this review, such as B. cephalus, C. macropomum, A. altiparanae, O. niloticus, and T. ovatus, the use of high stocking densities associated with the use of salt or anesthetic in the transport water has been efficient in mitigating stress for these species.

Conclusions
This review makes explicit the importance of the correct use of additives (salt and anesthetics) in water, as well as adequate stocking densities, during the transport of live fish.These factors can contribute to improving the well-being of most species by mitigating stress responses and promoting improvements in the quality of transport water, consequently enhancing the success of the entire fish production chain.Physiological and survival assessments post-transport and during the first days post-transport, as well as observations of return to feeding, are good indicators of response to evaluate the support of fish in the procedures adopted during transport.This review becomes an important tool for future studies, as it presents data that can be used for other species with phylogenetic similarities.

Table 1 .
Salt concentration recommended for fish transport (freshwater and diadromous fish are included).

Table 2 .
Synthetic and natural anesthetics recommended for fish transport (freshwater, marine, and diadromous fish are included).

Table 3 .
Stocking density recommended for fish transport.
* unit not specified in article.