Improving the survival rates of fish during and after transport is of interest to hobbyists and ornamental fish business alike. While hobbyists want their new acquisitions to arrive in good condition and to survive, they are also interested in keeping their cost down. Retailers have a need for quality fish and reducing post shipment losses to a minimum. At the same time, exporters seek ways to maintain water quality and control stress with the goal of reducing mortalities and enhancing profitability. Fish farmers and importers can also contribute along the way to everyone’s benefit. We all share a mutual interest in quality, healthy fish.

The ornamental fish industry has a standard for acceptable losses during air transport. Exporters are expected to compensate their customers when the dead on arrival (DOA) rate exceeds 5% (Lim, et. al, 2003). Please note that the overall losses of fish between capture through the first week post shipment is significantly if not unreasonably higher. This is why it is important that changes be implemented beginning with capture all the way to the marine aquarist. Each aspect of commercial ornamental fish enterprise should do their part to minimize these losses.

Hobbyists have an essential role in helping their stock to recover from the stress of capture, transport and handling. This begins with being informed. Learning and gaining knowledge about the hobby should be a priority. Research the compatibility, natural habitant and dietary needs of each specimen prior to purchase. To ensure the health of the fish it is advisable to wait until after having long-term success with “easy-to-keep” species before attempting more difficult or sensitive animals. Quarantine newly acquired fish for several weeks before placing them into the display aquarium so that the established stock is not at risk of contagious disease.

Packaging Protocol

The use of modern packaging technology to increase fish loading density and post shipment survival is critical to the industry. The accumulation of metabolic waste limits fish loading density. Several methods are available to reduce this build-up. These include starving the fish for a day or so prior to transport (Phillips & Brockway, 1954.), reducing water temperature (Lim & Chua, 1993. Teo & Chen, 1993.) and treating the fish with anesthetics (Chen, 1995b). Buffers (Amend et. al, 1982.) and drugs are often added to the transport water as well.

Fish are conditioned for packaging in three stages. These include prophylactic treatment, starvation and pre-packaging. The three stages of conditioning help reduce the amount of stress on the fish during transport, but they do not eliminate it.

Parasitic infection is common in ornamental fish. Infection may go undetected until the fish are weakened by the stress of transport and handling. Specimens weakened by disease or infection are less likely to survive transport stress. Prophylactic treatment (preventing or contributing to the prevention of disease) is usually performed on fish that may be infected with pathogens. However, the short duration of these treatments (1 to 7 days) may not be long enough to affect a cure.

Bacterial growth during transport produces ammonia, competes for the available oxygen supply and can potentially cause disease. Bacterial growth is controlled in transport water by the addition of dyes and antibiotics such as methylene blue, acriflavine and neomycin. .

Fish are inspected during the screening process for clinical signs of disease. These include external parasites, cloudy eyes, lack of appetite, dark body color, closed finnage and lethargy. Fish that exhibit indicators of disease or distress should not be packed for shipping. Four to six hours after pre- packaging the fish they should be checked again for signs of distress. Suspect fish should be removed from pre-packing to reduce the risk of mortality. Those fish can then be treated with the appropriate therapy. These fish can be re- evaluated, after allowing time for recovery, for future shipment.

Fish are starved for one to two days prior to shipment. This prevents the regurgitation of partially digested foods and reduces the volume of excreta during transport that would otherwise have a negative impact on water quality. Generally, small fish are starved one day and large fish for two.

Fish metabolism is three times higher than normal during transport (Froese, 1988). Ammonia and carbon dioxide are the two major metabolic wastes produced during shipping. Reducing the water temperature helps to minimize oxygen consumption and control the accumulation of acidity, carbon dioxide and ammonia in transport water. Typically, tropical species are shipped at 22C. In cold weather conditions, many exporters attach a heat pack to the inside of the lid before sealing to prevent a rapid drop in temperature. Cold packs may be used during warm weather conditions.

Some exporters use ammonia detoxifiers in the shipping bag. Ammonia detoxifiers can be incompatible with dyes such as Methylene blue and acriflavine. These dyes are commonly used as prophylactics in transport water. When the alkalinity of the water is not high enough, ammonia detoxifiers can cause a sudden drop in pH. To counteract this, a buffer that is compatible with the ammonia detoxifier should be used. A buffer mixture containing 80% sodium bicarbonate and 20% 5-mole borate is compatible.

Organic buffers such as Trizma™ or tris buffer may work best for maintaining the pH of water when fish density is high such as during shipping (Spotte, 1979). However, tris buffers are incompatible with some products used to control ammonia build-up. Tris is an amine and some ammonia detoxifiers react with ammonia and amines. Therefore, tris buffers should not be used in conjunction with these products.

Anesthetics are sometimes used to slow the metabolism of fish during transport. Tricaine or MS-222 is one of the most commonly used anesthetics. It is the only anesthetic fully licensed for use with fish (Stoskopf, 1993). Fish rapidly clear residues of MS-222, usually within 24 hours. However, the activity of MS-222 varies considerably with water quality, water hardness, fish species, fish size and fish density. This makes administering the correct dosage difficult.

The packaging process causes severe stress in fish (Barton & Iwama, 1991). Stress and the subsequent release of stress hormones, primarily cortisol, into the bloodstream suppresses immune function making fish more suceptible to pathogenic disease (Barton & Iwama, 1991). Acute, prolonged stress also causes osmotic or osmoregulatory dysfunction. If the fish are not able to recover normal homeostasis, it can lead to mortalities. Osmoregulatory dysfunction is counteracted by salinity manipulation.

The addition of salt to freshwater, or conversely reducing the salinity for marine species, is effective in controlling osmoregulatory dysfunction and other physiological responses to stress (Johnson & Metcalf, 1982. McDonald & Milligan, 1997). This will reduce mortalities caused by shipping stress (Tomasso et.al, 1980. McDonald & Milligan, 1997).

Fish are prepared for transport by packing them in polyethylene bags with little water. The cost of shipping limits the volume of water used in transport. The most important factor is an adequate supply of oxygen. The transport water is oversaturated with oxygen and usually pretreated with chemicals or drugs. Typically, the volume of oxygen to water in transport is three or four to one. The fish are then pre-packed and placed in an air- conditioned room so they can acclimate for 4 to 6 hours to a lower water temperature, confinement, crowding and high pressure prior to shipping.

Delayed Mortality Syndrome

Delayed Mortality Syndrome or DMS is associated with losses that occur in recently transported fish (Noga, 2000. Stoskopf, 1993). Fish suffering from DMS experience osmotic dysfunction and inhibited immune system response. Transport stress is diagnosed clinically by a decrease in plasma osmolarity in freshwater fish or an increase in osmolarity in saltwater species (Carmicheal et. al, 1984. Robertson et. al, 1988). Severely stressed fish can lose up to 10% of their body weight in 9 to 49 hours. This weight loss is attributed to osmotic dysfunction leading to dehydration (Sleet & Weber, 1982). These fish are susceptible to opportunistic pathogens, especially bacteria that take advantage of stress-weakened hosts (Mazeaud et. al, 1977). Infections caused by DMS usually become apparent within the first week after shipping. However, the critical period is longer extending to several weeks.

Steps to treat or reduce acute stress and DMS (Noga, 2000).

• Prophylactic treatment with antibiotics
• Frequent, small water changes.
• Reduce stress during transport and other manipulations such as netting.

Stress Management

The mean cumulative mortality at 7 days post shipment is approximately 11% (Lim, et. al, 2003). This does not include all losses that occur during the “chain of custody” between capture and final destination. Handling, capture and transport causes severe stress in fish making stress resistance an important factor in determining post shipment survival. Assuming that low stress resistance is partially responsible for mortalities that occur during and post shipment, the use of stress resistance techniques before transport may reduce mortalities. Current ornamental fish packaging focuses on minimizing stress and controlling the water quality during transport. More emphasis must be placed upon enhancing stress resistance prior to transport and post-shipment recovery of osmotic balance and normal homeostasis.

Fish are subjected to a series of stressors beginning with their capture in the wild or harvesting from a fish farm all the way to and including their final destination in a display aquarium. During collection for shipping, precautions should be taken to prevent injury, exposure to the air and overcrowding (Bartelme, 2003b). These stress factors can be more severe than the effects of transport (Iverson et.al, 1998). Netting and handling causes severe stress in fish, especially when they are removed from their native environment (water) during these processes (Bartelme 2003b, Carragher & Sumpter 1990, Klontz 1995, Kreiberg 1994, Rottmann et al 1992, Spotte 1993, Spotte 1979). Avoid using nets and exposing fish to the air during transfer whenever possible. Products such as StressGuard™ or Pro Tech Coat Marine™ can serve as a temporary barrier when the mucus/skin/scale barrier is compromised during handling and transport.

Significant portions of post shipment losses are due to osmoregulatory dysfunction and stress-mediated diseases occurring within the first week after transport (Johnson & Metcalf, 1982. Carmicheal et. al, 1984). Stress in fish causes osmoregulatory dysfunction (Harrell & Moline, 1992. Weirich et. al, 1992). This can lead to mortalities (Tomasso et. al, 1980). Reducing the gradient (difference in salinity) between the internal fluids of fish and the surrounding ambient water alleviates water and ion disturbance ((Wedemeyer, 1996). Manipulating the salinity of the transport water upward for freshwater fish and conversely downward for saltwater fish is effective for controlling osmoregulatory disturbances and reducing losses (Carneiro &Urbinati, 2001). Fish held in water that is close to isotonic (the salinity of the surrounding ambient water is close to the internal fluids of the fish) have increased stress resistance (Lim et. al, 2000). These fish also display a
significantly lower mortality rate at 7 days post shipment.

Marine teleost fish typically adjust to hyposaline conditions quite readily. An article in Drum and Croaker reported on thirty-two species of marine teleost fish maintained in a Specific Gravity of 1.010 for six to twelve weeks (Goodlett & Ichinotsubo, 1997). One study was performed using thirteen species of marine fish (Wu & Woo, 1983). Another was performed using Emperor angelfish Pomacanthus imperator. The angelfish were kept in salinities as low as 7ppt for 30 days without any apparent ill effects (Woo & Chung, 1995). While marine teleost fish adjust rapidly to salinities lower than natural seawater, the transition back from hyposaline conditions to NSW levels must take place slowly over several days. Fish with a different osmoregulatory strategy such as sharks and rayfish cannot withstand hyposaline conditions.

The optimal salinity for shipping saltwater fish has not been determined and may vary with the species. However, it is reasonable to assume that a salinity that is close to isotonic would work well for marine teleost fish. The cooperation of those at all points of handling is necessary. Ideally, marine teleost fish would be held in hyposaline conditions beginning with the exporter all the way to the hobbyist’s quarantine tank. This will help the fish quickly recover osmotic balance and it helps to control some types of external parasites as well. Hyposalinity therapy can be extended for several weeks as a proactive approach for dealing with some types of parasitic infection such as Cryptocaryon irritans (saltwater ich).

Acclimation to hyposaline conditions should begin two days prior to shipping. The fish should be acclimated in steps using two water changes per day for two days. Reduce the salinity 5ppt with each water change. I suggest a salinity of 14ppt, not to be confused with Specific Gravity, for transporting marine teleost fish. Hyposaline conditions should be maintained for 7 days or longer post shipment.

Maintaining the temperature of transport water during shipping is difficult. The temperature of transport water can rise or fall to dangerous levels leading to mortalities (Chow et. al, 1994b. Froese, 1998). Styrofoam boxes 2cm in thickness are generally used. It has been recommended that these boxes should be a minimum of 2.5cm thick (Froese, 1998) to reduce heat loss. .

Some exporters use buffers when transporting marine fish. Shipping fish in sealed plastic bags with oxygen can result in hypercapnia and mortalities unless the water is buffered to prevent a dangerous rise of carbon dioxide (Spotte, 1979). Organic buffers such as Tris buffer or Trizma™ do a better job of maintaining the pH than inorganic buffers such as NaHCO3 when the fish density is high (Spotte, 1979).

Fish that have been exposed to low pH will recover more quickly if they are acclimated slowly back to a normal pH. The pH of shipping water may drop below 7.0 so abruptly raising it into the 8.0 to 8.3 range is stressful to fish. This should be taken into consideration when acclimating fish into holding tanks. Raise the pH slowly over a period of a couple of days.

Do not float the shipping bags to equalize the temperature with the holding tank. The pH of transport water can rise quickly when the shipment bag is opened. This can lead to a rapid rise in toxic ammonia in the transport bag. Remove the fish quickly from the shipment bags into dimly lit holding tanks. The pH and temperature in the holding tank should be similar to that of the transport water. Then slowly adjust the water parameters to optimal levels. The salinity of the water should remain low for one week or longer.

Fish generally take much longer to adjust to changes in light intensity than humans do. Their eyes can take hours to adapt to a change from darkness to intense light. This makes it necessary to open the transport bags in dim lighting or with the use of red lights to avoid photo shock.

Nutritional Supplements

Enhancing stress resistance in fish should begin after capture at wholesale facilities or fish farms. Feeding fish diets supplemented with highly saturated fatty acids such as those found in Selcon™ helps with stress resistance. Supplementing the diet with vitamin C will improve disease resistance and help reduce the effects of stress (Waagbo et.al, 1993).

Enhancing Immune Function

Adding a biological immune defense modulator such as Beta 1, 3D glucan to the diet will help increase resistance to disease and stress. An overall enhancement of immune response can be achieved by the use of Beta 1, 3D glucan. It has proven in numerous scientific studies to be an immodulating agent that can enhance the major host defense mechanisms of the immune system (Bartelme, 2003c).

Pretreatment with Beta glucan means that the host can activate and proliferate defense mechanisms at a faster rate than invading organisms, giving the fish and their immune system a head start. Beta glucan can be administered in food prior to starving the fish in preparation for shipping. Safety evaluations indicate that Beta glucan is safe over a wide dose range, but a dose of 0.02% a day of body weight is recommended.

Beta glucan can be administered orally to fish by farmers, exporters, importers, retailers and hobbyists alike. It can be administered by exporters 24-48 hours prior to packaging. Importers and retailers can add Beta glucan to the food while the fish are in their care. Hobbyists can add Beta glucan to the food during the initial quarantine period before the fish are moved to their final destination.

Investigating Modern Packaging Technology

Stress from low pH is certainly a factor in transported marine fish. Although buffers such as tris-buffer are effective in stabilizing the pH of transport water, many exporters do not use them. There is concern about ammonia toxicity, especially when the pH is above 8.0. Other exporters claim that tris-buffer helps them to reduce losses. Sigma has a product called Trizma® “Fish” Grade Pre-set Crystal, pH 8.3, Type 8.3-FT, “Fish” grade, pH 8.3 designed to maintain the pH and control carbon dioxide toxicity in holding tanks and transport water. For more information about this go to http://www.sigmaaldrich.com/suite7/Brands/Sigma.html

Clinoptilolite (zeolite) is used to help control ammonia build up in freshwater during transport. However, it is not effective in saltwater. The use of ammonia detoxifiers can be problematic. They can be incompatible with dyes used to control pathogens in transport water. Ammonia detoxifiers can also cause a rapid drop in pH if the alkalinity of the water is too low. The use of ammonia detoxifiers and other means of controlling ammonia build-up in transport water warrant further investigation.

Breathing bags have been available for some time. These bags allow harmful levels of carbon dioxide to dissipate into the outside air. Despite some limitations and increased cost, they appear to have applications for use. New technologies for improving the practicality of breathing bags are being developed. For more information on breathing bags, go to http://www.novalek.com/klistfr.htm. (This is an example and not an endorsement of the product).

Notification Of Pertinent Information

Communication between the various points of handling is essential to improving the mortality rates of transported fish. Posting pertinent information in a standardized location such as on the outside of the styrofoam box on the lid will help foster this commuication. Some suggestions for information that can be shared are:

• What type of shipping bag was used?
• What type of buffer was used if any?
• What was added to control ammonia if anything?
• What is the salinity of the transport water?
• What chemicals or drugs were added to the shipping bag?
• Time and date that the fish were packaged for shipment.

Proposed Changes In Acclimation And Transport Procedure

• Hyposaline conditions in transport water and holding tanks for 7 days post shipment.
• Prophylactic use of Beta 1, 3D glucan 24-48 hours prior to shipping continuous through the first week post shipment.
• Notification of Pertinent Information.
• Styrofoam boxes 2.5cm in thickness.
• Further investigation into the use of tris-buffers, ammonia detoxifiers and breathing bags.

Concluding Remarks

It is crucial that scientific research continues to explore ways to reduce losses associated with transport and handling. We have much yet to learn. Many of the changes proposed here in procedures for transporting and acclimating fish will likely meet with resistance. Some may consider change to be inconvenient or be reluctant because of a potential increase in costs. However, if our hobby is to survive some tough choices must be made at this time. We cannot continue with the status quo. The responsibility to reduce fish mortalities belongs to everyone involved. After all, we share a mutual interest in quality, healthy fish.

References

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