 The purpose of this video is to acquaint you, the viewer, with the techniques of hormone-induced spawning of fish. We recommend that references available from the Southern Regional Aquaculture Center, the University of Florida's Department of Fisheries and Aquaculture, as well as other agencies, should be studied for the requirements for each species of fish. The video presents a considerable amount of information, too much to be assimilated in one viewing. We suggest that you view it several times, using the pause and review options of your VCR for maximum benefit. Hormone-induced spawning of fish. Hormone-induced spawning can be used for large sport fish as well as tiny ornamental aquarium fish. Spawning manuals are available for many types of fish. However, the procedures for mixing hormones, sampling the ovaries, determining the stage of maturity of the eggs, injecting the fish, and taking the spawn by hand stripping or tank spawning are similar for widely differing species. Hormone-induced spawning has been successful for the entire range of fish from the primitive paddle fish and sturgeon to carp, striped bass, mullet, snook, and red drum. Techniques used on one species of fish can be adapted with only minor modifications to other species. Many desirable species of fish spawn in tidal estuaries and large rivers. These spawning environments are nearly impossible to simulate in a hatchery. Hormone-induced spawning is the only alternative for successful hatchery breeding of these fish. First, let's look at a simplified explanation of the fish's internal control mechanism that triggers spawning. The brain receives and processes information such as water quality, light, and temperature. Those factors that are important for reproduction stimulate the brain to produce a releasing hormone that causes the pituitary, a small gland located beneath the brain, to produce sex hormones. These sex hormones act on the ovaries, causing them to develop and release mature eggs. Cart pituitary is the most common hormone used for induced spawning. It has been shown to be effective on a wide variety of fish species and is readily available from commercial suppliers. A mortar and pestle or hand tissue grinder is used to pulverize the pituitary material. Bacteriostatic water should be used when mixing cart pituitary to minimize bacterial contamination. The liquid is added a little at a time, mixing thoroughly to produce a uniform suspension. The hormone requires 20 to 30 minutes to go into solution. Cart pituitary also contains the pituitary tissue as well as other hormones not directly involved in reproduction. The tissue residue should be allowed to settle to the bottom of the vials. Only the liquid above the tissue residue is injected. Cart pituitary is injected at a dosage of 2 to 8 milligrams of dried powder for each kilogram of fish body weight. Purified hormones are frequently used for induced spawning. The most common of these is human chorionic gonadotropin, or more simply HCG. HCG is measured in international units. Injections of 165 to 600 international units per kilogram of fish body weight are usually recommended. HCG has been used in combination with cart pituitary for grass carp and the red-tailed black shark, a popular aquarium fish. The combination has been shown to have greater potency than either preparation used alone. Lutinizing hormone-releasing hormone, or LHRH, is produced by the brain. It stimulates the pituitary to produce and release sex hormones. Synthetic analogs of LHRH, referred to as LHRHA, have been manufactured commercially. These synthetic analogs are not derived from animals but are produced in a laboratory. In addition, modifications have been made in the protein chain. As a result, these hormones are more potent than the fish's own releasing hormone. LHRHA is injected at 5 to 10 micrograms for each kilogram of fish body weight. A microgram is one one-thousandth of a milligram. Dopamine is also produced by the brain and it controls the release of sex hormones from the pituitary. Unlike LHRH, dopamine inhibits the release of hormones from the pituitary. There is a family of drugs that act as dopamine blockers, either by preventing the release or by inhibiting the absorption of dopamine. Some fish, such as the red-tailed black shark, do not respond to injections of LHRHA alone. The use of dopamine blockers, such as reserpene and haloperidol, greatly increase the effectiveness of LHRHA for difficult to spawn species. The recommended dosage for reserpene is 10 to 50 milligrams per kilogram of fish body weight. Haloperidol is injected at one-tenth to one milligram per kilogram of fish body weight. Hormones, such as HCG and cartpituitary, bypass the brain pituitary link, acting directly on the ovaries. LHRHA, on the other hand, stimulates the fish's own pituitary to produce and release sex hormones needed for spawning. However, if the fish's brain perceives that conditions are not optimum for spawning, dopamine is produced. This inhibits the release of sex hormones from the pituitary. Dopamine blockers, such as reserpene and haloperidol, prevent this negative feedback. Enhancing the Infectiveness of LHRHA. The volume of the injection is measured in cubic centimeters, commonly referred to as CCs. Injections should be small enough to avoid injuring the fish, yet large enough to be accurately measured. To calculate the concentration of hormone to be mixed with a vial of sterile water, the recommended dosage, multiplied by the approximate weight of the brewed fish, is divided by the desired volume of the injection. For example, if the dosage is 4 milligrams per kilogram, the fish weigh approximately 10 kilograms, the desired volume of injection is 1.5 cc, and the volume of a vial of sterile water is 10 cc, then the concentration of hormone is equal to 4 milligrams per kilogram, multiplied by 10 kilograms, divided by 1.5 cc, in this case, 30 milligrams per cc. The quantity of hormone to be mixed with the vial of bacteriostatic water is calculated by multiplying the hormone concentration, 30 milligrams per cc, by the volume of the vial, 10 cc. 300 milligrams of hormone are then weighed and mixed in a 10 cc vial of water. HCG and LHRHA come in a predetermined quantity in a sterile vial. The volume of sterile water that must be added to the vial must be calculated to obtain the desired concentration of hormone. For example, if you have a 5 milligram vial of LHRHA, the recommended dosage is 10 micrograms per kilogram, the fish weigh approximately 50 grams, and the desired volume of injection is 1 tenth of a cc, then the desired concentration of hormone is equal to 10 micrograms per kilogram, multiplied by 50 grams, or 0.05 kilograms, divided by 0.1 cc, in this case, 5 micrograms per cc. The volume of water that must be mixed with the vial to get the desired concentration is equal to the quantity of hormone in the vial, divided by the desired concentration. This equals 1,000 cc. Rather than mixing such a large volume of hormone, three dilutions are made. This can be accomplished by first drawing the sterile water from a 10 cc vial and mixing the contents with the vial of LHRHA. 9 cc's of this mixture are then placed in sterile 1 cc vials and labeled 500 micrograms per cc, or 0.5 milligrams per cc. The remaining cc of LHRHA is mixed with 9 cc's of sterile water. 9 cc's of this mixture are then placed in sterile 1 cc vials and labeled 50 micrograms per cc, or 0.05 milligrams per cc. Once again, the remaining cc of LHRHA is mixed with 9 cc's of sterile water. 10 cc's of this mixture are then placed in sterile 1 cc vials and labeled 5 micrograms per cc, or 0.005 milligrams per cc. All hormone preparations must be properly labeled and stored in the freezer so that only the required amount need be defrosted, saving the potency of the remaining vials. Using sterile procedures, hormones can be stored in a freezer for several years without loss of potency. The following items are needed to sample the eggs in the ovary to determine if the brewed fish are ready to spawn. Sampling the ovary is the only method presently available to accurately determine the stage of maturity. The eggs must be in the final stages of development before hormone spawning can be successful. Before sampling, the fish are quieted with an anesthetic. Brewed fish must be handled carefully to minimize physical injury and stress. The importance of this point cannot be overemphasized. Cumulative physical stress to the fish can have a greater detrimental effect on spawning success than almost any other factor. MS triple 2 is presently the only anesthetic approved for food fish. The external appearance of the female, such as plumpness of the abdomen and redness of the vent, has long been used to evaluate maturity. However, these characteristics are extremely subjective and can be misleading. The ovaries of most fish can be sampled with either a rigid or flexible tube of appropriate diameter. The tube is inserted through the genital opening into the ovary. A small amount of section may be applied, if necessary, to draw a small number of eggs into the tube. The diameter and color of the egg is a good indicator of development. The primitive egg cells are very small, hardly larger than other cells in the fish. In many species, they are nearly clear. With the onset of yolk formation, the primitive egg cells become eggs. During this stage, the eggs enlarge substantially and yolk material is deposited, causing the eggs to appear opaque. The color varies with the species of fish. At the completion of yolk formation, the eggs are considered ripe. However, additional development is necessary before they can be fertilized. As development continues towards ovulation, the nucleus moves from the center of the egg to one end. Observing the position of the nucleus is an excellent method of determining egg development. The eggs may be cleared by placing them in a solution of 60% ethanol, 30% formalin, and 10% glacial acetic acid by volume. For many species, the nucleus can be observed with a microscope by simply placing a cover slip over the eggs on a glass slide. The nucleus appears as a translucent circle in the egg. Immature eggs appear smaller and are nearly clear as compared to ripe eggs. In addition, the nucleus is located in the center of the egg. As the egg matures, the nucleus moves from the center to the edge of the egg. This signifies that the eggs are sufficiently advanced so that hormone-induced spawning can be successful. Eggs that have begun to break down in the ovary appear whitish in color. In addition, the egg contents are irregular in composition and appear to have pulled away from the shell. To minimize stress, it is preferable to estimate the weight of the brewed females rather than inflicting damage to the fish by weighing them in a bag from a hanging scale or laying them on a platform scale. To determine the volume of hormone to be injected, check the literature for the recommended dosage for the fish species. The total volume of hormone to be injected can be calculated by multiplying the recommended dosage by the weight of the fish and then dividing by the concentration of the hormone mixture prepared earlier. For example, if the dosage is 4 mg per kg, the fish weighs 6 kg, and the concentration of hormone is 30 mg per cc, the fish is injected with 8 tenths of a cc. The number of injections is dependent on the response of each species to the selected hormone. Some species ovulate following a single injection of the total dosage. However, multiple injections are usually more successful. A priming dose of 10 to 20% of the total dosage is administered to advance egg development, and the resolving dose stimulates ovulation. Males are usually given only a single injection. Injections should be scheduled so that the time of induced ovulation coincides with the fish's natural spawning period. Hormones are usually injected intramuscular rather than intraparitoneal because it results in a more constant delivery of hormone and there is less chance of injuring the fish. The preferred site for an intramuscular injection is into the thick muscle of the back. The needle is inserted directly behind the dorsal or back fin where there are no scales. When injecting the fish, every effort must be made to minimize stress. Fish may be quieted with an anesthetic such as MS triple 2. Do not remove large fish from the water when giving injections. The tank spawning method is the simplest way to obtain the spawn from fish that have non-sticky, floating or semi-boyant eggs. A round tank is advantageous for spawning riverine and estuarine fish because the circular flow simulates the current in which these fish naturally spawn. The drain water from the tank empties into a screened container. Brood fish of both sexes are placed into the spawning tank. The female ovulates when she is physiologically ready. The males stimulate the female to release the eggs in the water and fertilize the spawn. The non-adhesive eggs are carried with the water flow from the spawning tank and collected in a screened container. The eggs are then transferred to an incubator. The advantages of tank spawning are that it is unnecessary to predict the exact time of ovulation so over-ripening of eggs in the ovary is not a problem. There is less potential of injury to the brood fish and less labor is required. The disadvantage is that tank spawning cannot be used for fish with sticky eggs or when special treatment of the eggs is necessary for induction of polypoiety. Tank spawning is especially useful for large, delicate sport fish. The most common method used for taking the spawn is hand stripping and dry fertilization. Prior to the earliest anticipated time of spawning, the female fish are captured and checked to verify ovulation. Gentle finger pressure is applied to the abdomen starting at the pectoral fins and slowly moving toward the vent. Do not try to squeeze or force the eggs from the fish. This will only injure the female. Frequent or rough handling of female brood fish retards ovulation, reduces success and increases fish mortality. When eggs flow freely from the vent, the fish is dried with the towel and gentle pressure is applied to strip the eggs into a dry bowl. It is important to ensure that no water comes in contact with the eggs until after the milk is added and mixed. Water activates the sperm and causes the micropile of the egg, the opening through which the sperm enters the egg, to close. Knowledge of the anticipated time of ovulation is of utmost importance if you intend to hand strip the fish. The time between injection and ovulation is controlled by the species of fish, the water temperature, the hormone used, and the stage of development. During ovulation, the blood connection between the female fish and the eggs in the ovary is eliminated. The eggs become oxygen starved and their quality can deteriorate rapidly. In general, the eggs of tropical and subtropical fish become overripe earlier than those that spawn at temperate and cool water temperatures. Check the literature to find the maximum grace period between ovulation and overriping of the eggs for the species you are spawning. The milk can be obtained by the same technique used for stripping the eggs. The male fish is dried and the milk is stripped onto the eggs. Tremendous number of sperm are present and only a drop of milk. The eggs and milk are gently mixed and a small amount of water is added to activate the sperm, fertilizing the spawn. Sperm is usually modal for only one minute or less. Milk usually appears creamy white in color and consistency. When available, milk from two or more males is used to ensure fertilization of the spawn. Individual males can be used to fertilize more than one spawn. Milk can also be collected prior to stripping the eggs. The milk is stripped and immediately drawn into a dry sterile syringe. The milk is then expelled into a small plastic bag. Antibiotics can be added to inhibit bacterial development and the bag is filled with oxygen and stored on ice in a refrigerator. Do not freeze the milk because this will kill the sperm. Adhesive eggs should be treated at this time with substances such as silt, tannic acid or urea and salt to deactivate the sticky layer. Initially, the developing embryos of some species are sensitive to movement. Compressed oxygen is added to maintain water quality during this critical stage. The fertilized eggs can then be transferred to the incubation apparatus. The purpose of the incubator is to provide ideal conditions for the developing embryos. A wide variety of incubators have been used. The McDonald jar, however, has been the traditional hatching apparatus for almost every food and sport fish species produced in the United States during the past century. Conical baskets, conical vats, circular tanks and paddle troughs have also been used to incubate the eggs of various species of fish. Air-aided water is delivered to the jars through a low-pressure manifold. The optimum water quality and temperature and the rate of development of the embryos varies with the species of fish. Water is delivered through a tube to the bottom of the jar. The water current produces an upwelling that keeps the eggs in suspension and constantly oxygenated until they hatch. Although the spawning process is completed, the hatchery work has just begun. Now, however, is the time to analyze your failures and learn from your mistakes. Were the brewed fish in proper spawning condition? Did you encounter water quality problems that could have been prevented? Was the hormone mixed and stored properly? Was the hormone dosage calculated correctly? Did handling stress and or disease kill or impair the brewed fish or larvae? Did improper timing and taking the eggs result in poor egg quality? Did sloppy handling of the spawn reduce the percent fertilization? Did equipment failure result in death of brewed fish, embryos, or larvae? Induced hatchery spawning is a fairly complicated process requiring facilities, hard work, and strict attention to detail. The process starts long before the spawning season with the preparation of facilities and the conditioning of brewed fish. There are many ways to fail at each step. So before initiating an aquaculture venture, learn as much as possible from videos, books, extension publications, journal articles, established fish farmers, state and federal hatchery workers, and extension personnel. Lessons learned in this way are much less painful than those punctuated by dead fish and lost revenues.