WO2005039280A1

WO2005039280A1 - Process for transport of live fish without water

Info

Publication number: WO2005039280A1
Application number: PCT/PH2004/000008
Authority: WO
Inventors: Bonifacio F. Comandante, Jr.
Original assignee: Comandante Bonifacio F Jr
Priority date: 2003-10-24
Filing date: 2004-09-16
Publication date: 2005-05-06

Abstract

Conditioning techniques and temperature manipulations were done on fishes to arrive at optimum parameters that would allow live fish transport without water. Normal aquatic transport procedures wee done at different temperatures and similar treatments were done on fishes in waterless conditions. Fish sourcing, material preparations and collection of water samples were done prior to transport experiments. Individual fish experiments for non-aquatic conditions were done at room temperatures while loading density experiments were performed at 25 °C to simulate plane transport. Low temperature and zero pressure may have prevented the collapse of fish gills, thus enabling direct assimilation of pure oxygen due to a temperature gradient between fish gills and its external environment. The process cited above presents a new method for transporting fishes. Current methods of fish transport utilize water which is messy and costly. This new technique will provide tremendous savings as well as increase the survival of fishes during transport.

Description

PROCESS FOR TRANSPORT OF LIVE FISH WITHOUT WATER BACKGROUND OF THE INVENTION Finfish cargo forwarding in non-aquatic conditions or without water presents a paradigm, shift for the live food fish trade. In December 2003, the pioneering technology was successfully presented and defended among a panel of scientists -from Silliman University Marine Laboratory. The panel included Dr. Hilconida Calumpong, Director of SUML, Dr. Janet Estacion as thesis adviser and four Professors from Silliman University. Dr. Angel Alcala was also consulted several times during the course of the study. In addition, Dr. Ricardo del Rosario of the Institute of Food Science and Technology, UPLB and Dr. Dan Lindstrom of the University of Hawaii provided valuable insights to the technological breakthrough. Live fishes command a premium price because of its guaranteed freshness, intrinsic flesh characteristics, quality and delicate flavor in comparison with its chilled counterpart. Tastes, cultural traditions and cooking styles (mostly Chinese in origin) determine the live fish market requirements. The consumption of live fish is a niche market for affluent tourists, the upper class population and businessmen. With the gradual recovery of world economy, a positive trend in the demand for high-value live marine and freshwater finfish is expected. The aim therefore of live fish transport without water is to provide its new technology to the global aquaculture industry with emphasis on export destinations like China, Hong Kong, Japan and Chinatown communities worldwide.

BASIS OF THE INVENTION: THEORETICAL FRAMEWORK The present invention of the live fish transport without water presents a paradigm shift in manipulating fishes and its environment. When the fish leaves its water environment, it must have with it an enclosed environment that will supply necessary vital needs. During transport, live fish are placed in a completely self sustaining artificial ecosystem, with sufficient oxygen stored and where carbon dioxide and other waste products can dissipate/disperse within the enclosure. In transport, without water, fish will have to adapt to the new environmental problems such as waterlessness and negligible pressure. Tranquilizers and various chemical agents are often used to sedate live fish in preparation for transport. One of the most commonly used chemical anesthetic is Tricaine Methanesulfonate (Subasinghe 1997). One model in transporting marine organisms in non-aquatic condition using hibernation techniques is that of prawns and lobsters (Subasinghe 1997). Harvested prawns or lobsters are transferred to tanks with sufficient aeration. Water is slowly lowered by adding small pieces of ice in plastic pouches. Several factors such as place of origin of the animal, season and age influence the hibernation temperature. Temperature of the cooling tank should be dropped in stages to 08-12°C or even lower for animals from colder environments. Cooling at this temperature for 20-30 minutes induces hibernation (Subasinghe 1997). Oxygen availability during fish transport is one critical factor which has to be addressed properly, to prevent anaerobiosis which can occur at concentrations of lower than 2.5 mg 02 li -1 (Woo and Wu, 1984). Current practice utilizes medical grade oxygen pumped in plastic bags containing water and fishes. This practice enables transporting of fishes even up to 30 hrs. (with water) Temperature, another critical factor, plays an important role in oxygen uptake. Gill water permeability is temperature sensitive (Loretz 1979) and there is lower osmotic permeability of fish gills at high temperature (Robertson and Hazel, 1999). Oxygen and temperature synergism present conditions whereby continuous respiration of the organism takes place. The amplified surface areas of fish gills and the blood-to-water diffusion are critical to gas and osmotic water exchange. Fish gills are characterized by an expansive epithelial surface in direct contact with the external environment (Hughes and Morgan, 1973). On the other hand, apical plasma membranes of epithelial cells represent the main gill-environment barrier membrane (Sardet et al, 1979). Water quality can limit productivity in culture systems (Chiba 1986) and maintaining good conditions during transport are important for survival and subsequent recovery and growth. Dissolved oxygen (DO) is a critical parameter to be monitored regularly in all phases of fish culture and more importantly during transport. Temperature, another critical parameter, deserves some critical degree of monitoring. Other factors such as inorganic nitrogen and salinity to some extent are factors critical for fish survival during longer transport periods. In fishes, gill water permeability was found to be temperature sensitive (Loretz 1979). Cellular membranes of fishes undergo compensatory lipid compositional changes during thermal acclimation (Hazel and Williams 1990) such that influence of membrane lipid composition on the osmotic permeability of teleost gills showed that without calcium, trout gill osmotic water uptake values increased 1.5 to 2 fold. Robertson and Hazel (1999) studied the influence of temperature on the osmotic water uptake of rainbow trout (Oncorhynchus mykiss) and nile tilapia (Oreochromis niloticus). Both species were housed in fresh water recirculating aquaculture systems. Water temperatures were controlled using in-tank chiller or heating units. Trouts were acclimatized at 5°C and 20°C while tilapia at 21.5°C and 33°C. Results with both trout and tilapia indicated that water moved osmotically into isolated, ligated gills in an acutely temperature sensitive manner, consistent with models of membrane water permeation. Highest initial rate and extent of water uptake were observed in gills from lower temperature acclimated trout or tilapia The markedly low osmotic permeability of tilapia gills at 33°C is also consistent with a stabilizing membrane compositional response associated with acclimation to high temperature. There are different water management practices that can influence water quality. The dynamics of artificial aeration and circulation of water in brackish shrimp ponds showed that dissolved oxygen concentration was highest with continuous aeration while ammonia concentrations were at its lowest levels, during the same period (Sanares et al, 1986). High density of fishes practiced during transport is faced with problems of water quality. Toxic metabolites such as un-ionized ammonia and carbon dioxide are released and accumulate in the water medium Anrnionia, the waste product of protein metabolism, is toxic in high amounts (Begarinao 1991). In an aquatic environment, the manner by which water parses through biological membranes of gills and the physiological implications of such process remain important issues in cellular and organismal biology (Robertson and Hazel, 1999). The concept that lipid bilayers can act as barriers to water permeation in biological systems is supported by the recent identification of protein channels or aquaporins dedicated to trans-membrane water transport (Preston et al, 1992; Verkman et al, 1996). Fish gills are characterized by an expansive epithelial surface in direct contact with the external environment (Hughes and Morgan, 1973). The fish gills amplify surface area, the blood-to-water diffusion within the same area is critical to gas fexc uige. #nd osmotic water exchange. This is due to the differences in gradient across the gill membrane and the outside environment. In freshwater trout, the critical interfacial barrier is predominantly 96% composed of gill epithelial cells (Isaia 1984 in Robertson and H z^l, 1999). Ijnpermeable junctions link these cells (Sardet et al, 1979) wijile apiςal asma membranes of epithelial cells represent the main gill-environment barrier membrane. Robertson and Hazel (1999) in their earlier

udied the influence of membrane lipid composition on the osmotic ferrøgaj jlity of teleost gills showed that without calcium, trout gill osmotic water, upta e values increased 1.5 to 2 fold. The specific contribution of cholesterol to restricting barrier membrane water permeability was indicated by concentration - dependent increases in water uptake in the presence of pore-forming agents like nystatin or methyl-β-cydodextrin. In addition, a cholesterol-specific cytochemical probe (filipin) intensely labeled the apical surface membranes of trout and tilapia gill epithelium. In summary, the studies implicate membrane cholesterol in determining water permeability in fish gills.

EXPERIMENTS DONE TO ARRIVE AT INVENTION The specific objective of experiment is to determine fish survival, weight changes and behavior in non-aquatic condition at varying starting temperatures. A total of 36 observation boxes containing one fish per box were prepared. Each box representing a replicate had two double glass slots to serve as windows for observations. Styrofoam boxes were used to maintain desired temperatures to which the fish were exposed to. Standard plastic bags

(grade 003, 25" x 30") were used to enclose the fish. These plastic bags can hold approximately 0.05 m3 (2.5g Oxygen) of hospital grade oxygen when inflated. The rule of thumb ratio 1:4 was used i.e., 1 kg fish: 4 kg water.

Fishes should be conditioned in enclosed containers intermittently prior to actual transport. Fishes originating from net cages can be transported immediately.

Preparation of Fish Chemicals Transport of large and spiny fishes require the use of anesthetics to prolong the state of low metabolism or activity. This minimizes oxygen uptake and prevent jerking or sudden motion which accounts for punctured plastic bags, thus loss of oxygen during transport. Two most common commercially available anesthetic in the market are quinaldine (2-4 methyl chinolin) and MS222 (Tricaine Methanesulfonate). These require great care in handling for health and safety considerations. Both anesthetics require fish holding period prior to human consumption after treatment (Subasinghe 1999). Methanesulfonate or MS222 requires 30 days holding period before the fish are used. Another anesthetic (Aqui-S) developed in Upper Mississippi Research Center is claimed by the sponsor to be zero withdrawal time (Aquaculture Magazine 1999). Unfortunately, it is not available in the Philippines. A seawater chemical recipe was formulated and calibrated vis-a-vis MS222 to produce similar sedative effects. Experiments on use of sea water anesthetic recipe in combination with different temperature regimes were performed. Pre-transport Conditioning For pre-transport conditioning, fishes were starved for 48 hours inside concrete water tanks. Afterwards, they were transferred to recirculating holding tanks containing 30 li of saline water at 18 ppt. From an initial temperature of 26°C, water temperature was lowered down by 4°C every hour until 18°C was attained (500 grams of ice will lower temperature of 10 li saline water in 20-30 minutes). Anesthetic was added to fresh water in holding tanks to provide a salinity of 18 ppt and fishes were individually exposed to the anesthetic bath for 5 min prior to transfer to plastic polyethylene bags. Random sampling procedures were utilized in placing fishes at pre-numbered styrofoam boxes. Weighing of fishes Individual fishes were weighed directly using Yamato lOOOg (graduation division, d = 5g) scale. Excess water was removed by placing plastic tray on dry cloth prior to actual weighing procedure. Extra caution was taken to prevent too much stress to the fishes at this stage by preventing unnecessary movements and exposure outside the plastic bags. Immediately after weighing, fishes were transferred to plastic bags without water. A cloth (18 cm x 30 cm) was soaked in the appropriate experimental temperature to transfer the desired pre-transport coldness inside the plastic bag. Said bags were sealed with elastic rubber bands after introducing 0.05 cu. meter of hospital grade oxygen. Four pieces of ice in plastic bags (8 cm x 30 cm) were placed inside the styrobox. Monitoring of Physical Parameters Hourly readings of temperature at the bottom portion of the plastic bags and the styrobox ambient temperature were recorded. Holes were made at the top and lower portions of the styrobox to accommodate the insertion of laboratory thermometers. Salinity of conditioning transport water was determined using

ATAGO S-10E refractometer. Fish Observations Hourly readings of opercular movement were recorded while fish behavior was determined by digital video camera every two hours. Motion of operculum per minute was counted for each fish. Similarly, fish behavior was recorded specifically focusing on the position of dorsal, pectoral and caudal fins. Attempts were made to record fish color, eye movement and body spots. The absence of a consistent benchmark to note changes in color, eye movement and spots prevented any reliable recordable observation. Fish survival data were initially attributed to opercular movement observations for the first 12 hrs. Succeeding observations were made in water recirculating tanks based on 24, 48 and 72 hrs durations.

PREPARATIONS FOR LIVE FISH TRANSPORT WITHOUT WATER Transporting live fish to markets sometimes several hundred kilometers away is stressful to fish and deserves special attention. Fish is conditioned prior to transport over long distances to reduce stress. This is done by starving the fish 48 hrs before harvest. Live reef fish can be given a freshwater bath for 2-3 minutes when they are first delivered to the transport staging center (freshwater kills various external parasites). Harvesting is done by lifting the cage nets or draining ponds and scooping a small number of fishes each time to prevent damage. The fishes are then transported by small boats or trucks to packaging centers. Carrier boats of live fish are equipped with small water pumps and aerators. Live fish collected from culture cages are transferred manually to seawater tanks with adequate supply of oxygen. Fishes originating from ponds are transported by trucks. Features of live marine land transportation system are as follow: ♦> Truck: A five- ton payload flatbed truck equipped with seawater tanks (with aeration fittings, air distributors/dif-users and water drainage) ❖ Small water pump ❖ Aircon compressors to provide oxygen (powered by truck's engine) ❖ Scoop nets (stainless steel frames and nylon netting) ♦> Weighing baskets In aquatic transport condition, decline in water quality is the biggest problem i.e. lowering of oxygen content, carbon-dioxide build-up, detrimental changes in pH and build-up of fish wastes. These problems are non-existent in waterless transport. Every minute counts when packaging live fish for air transport. An experienced operator knows how much time his team needs to work on a live fish styrobox and plans so that the group can start packaging with just enough time for them to finish their task prior to airport cargo check-in. Ideally the packaging center should be no more than a 30-minute drive from the airport. In terms of seawater requirements, costs can be reduced if seawater can be directly pumped to the packaging center when required. This also cuts down the cost of water holding facilities, which are essential for any packaging center. An immediate water supply is needed for when the water in the holding tanks suddenly turns bad. Whereas before, water was used for shipment, it will now be limited to holding fishes in tanks. The temperature of the holding water should be in the range of 21- 23°C. Since reef fish typically live in water ranging at temperature from about 24 to 30°C, gradual reduction in temperature will render the fish less active and less stressed. The water in the holding tanks should be well circulated and must go through a sand/biological filter to neutralize fish wastes. Injecting air using an air compressor and diftuser can raise oxygen content. The water temperature should be lowered by another 2-3 °C prior to packing. The chilling process should be slow and completed within two hours. The limited and precise timing of packaging live fish in polyethylene0 plastic bags requires a skillfid working team. Fishes are placed in double plastic bags inside a styrobox. The box is passed on to a second worker who is responsible for the injection of pure oxygen and closing the bag with an elastic band. It is then passed on to the final worker who checks the packing, adds coolant, and seals the styrofoam box with an adhesive tape. An5 experienced working team can process one box in 60 seconds. Every minute counts when packaging live fish for air transport. An airplane pallet can accommodate 126 styroboxes which is equivalent to 450 kg of live fish. An experienced team will need 30 min to pack the said volume. The basic time constraint when the fish is in waterless condition is0 summarized below: Time Requirements for various fish transport activities Activity Duration (hrs) Remarks Batch packing 0.5 Good for 126 styroboxes or one pallet Travel to airport 0.5 Packaging area must be within 30 mins radius to airport. Customs and waiting time 2.0 Flights cannot af ord delays of more than two hours (retrieval becomes a must) Flying time 5.0 Maximum flying time to be considered. Customs Release 1.0 When time is maximized, there is a need to put fishes in water after customs release.

The precise timing necessitates that any air transport staging center is a Hazard Analysis and Critical Control Points (HACCP) certified facility. BRIEF DESCRIPTION OF THE DRAWING FIGURE

The drawing figure illustrates the components of the present invention

DETAILED DESCRIPTION OF THE DRAWING FIGURE

In the drawing FIGURE, as shown by the exploded perspective, artificial ecosystem support system 1 and 4 are depicted with a styrobox 2 and its cover 6. Fishes 3 are illustrated by an elastic rubber band 5. As illustrated, the plastic 1 is inflated by pure medical oxygen 4 containing the live fishes. The enclosing plastic bag has a thickness of 003 suitable for rough movement of the fish in some instances. In accordance with the invention, the styrobox 5 and the cover 6 enables the maintenance of transport temperature critical for fish survival. The plastic 1 is scaled with elastic bands. Finally, medical oxygen 4 is impregnated to the plastic bag to enable direct contact of oxygen to the fish gills. Examples As indicated earlier, the following examples further illustrate the present invention. Example 1 36 specimens of the species Epinephalus tauvina or brown groupers have been experimented to test survival, weight changes and fish behavior over 9 hours in non-aquatic conditions or without water and another 72 hrs in water tanks.

Claims

The results can be seen in the table below: Results of Verification Experiment

Example 2 72 specimen of the species Epinephalus tauvina or brown groupers have been tested in 12-boxes for different loading densities. Survival and weight changes were determined after 9 hrs in non-aquatic conditions or without water and another 72 hrs in water tanks. The result can be seen in the table below: Summary of Fish Survival (%) in Non-aquatic Condition for Different 10 Loading Densities.

INVENTOR'S CLAIM 1. A process for transport of live fish in non-aquatic condition or without water brought about by (i) fish conditioning in water tanks (ii) temperature manipulations to desired levels that induces fish hibernation or the state of very low metabolic activity and (iii) packaging in plastic polyethylene bags without water (iv) impregnated with pure medical oxygen (v) sealed with double elastic rubber bands 5 and (vi) maintained in desired ambient temperature inside Styrofoam boxes.

2. A process according to claim 1 wherein the live fishes are both marine and freshwater species.

3. A process according to claim 2 wherein the species used are for food o and aquarium trade purposes.

4. A process according to claim 3 wherein species include vertebrates and invertebrates.

5. A process according to claim 1 wherein temperature manipulations are done to induce organism hibernation or the state of low metabolic5 activity.

6. A process according to claim 1 wherein plastic polyethylene plastics or suitable containers are used devoid of water as a means for oxygen uptake by gills.

7. A process according to claim 1 where the absence of water does not0 inhibit oxygen uptake by fishes.

8. A process according to claim 7 where medical oxygen is directly assimilated by animal gills or respiratory organs.

9. A process according to claim 8 where medical oxygen can be commercially prepared or artificial reactions from Hydrogen Peroxide.5

10. A process according to claim 1 further comprising maintaining the package environment at a temperature range of from -4°C to +35°C.

11. A process according to claim 1 wherein plastic polyethylene bags with medical oxygen are sealed with elastic rubber bands.

12. A process according to claim 1 wherein loading densities of fishes are sufficient to the ratio of introduced medical oxygen which is about 5 grams or 0.1 m³.

13. A process according to claim 1 wherein the fishes are either cultured or caught from the wild.

14. A process according to claim 1 wherein the fishes' weight ranges from 5 grams to 50 kilos or more.

15. A process according to claim 1 wherein fish conditioning are done prior to harvesting and during pre-transport activities.

16. A process according to claim 15 wherein fishes are starved and evacuated prior to transport.

17. Packaged fishes undergo a waterbath treatment (i) containing anti- stress chemicals at (ii) varying temperature regimes (iii) for certain time durations. Conditioning techniques are done using waterbath with anti-stress chemicals to prevent skin infections and induce very low metabolic activity. Different temperatures are used to manipulate desired fish activity. The time duration varies from the size of fishes as well as the species.

18. Packaged fishes according to claim 17 are dipped in a specifically prepared waterbath.

19. Packaged fishes according to claim 18 reacts to the synergism of temperature changes and chemical dosage.

20. Packaged fishes according to claim 17 wherein temperature exposures are dictated by the fish size and species.

21. Packaged fishes according to claim 17wherein fishes are prepared to waterless transport by protection offish mucous.

22. Packaged fishes according to claim 17 wherein fishes are induced to hibernation or very low metabolic activity.

23. Packaged fishes according to claim 17 wherein different temperatures are utilized to prevent hyperactivity.

24. Packaged fishes according to claim 23 wherein use of temperature adjustments vary from fish to fish sizes as well as conditions of gill cover or operculum.

25. Packaged fishes according to claim 23 wherein temperature exposure duration are dictated by the desired waterless transport period.