Techniques for Taking and Fertilizing the Spawn of Fish

SRAC Publication No. 426

Southern Regional Aquaculture Center

November 1991

R.W. Rottmann, J.V. Shireman, and F.A. Chapman*

Following hormone injection, the During ovulation, the connection tion of egg quality for some eggs and milt of fish can be taken between the female fish and the species of fish commonly spawned by several different methods: eggs in the ovary is eliminated. In by hormone injection. warm-water fishes, egg quality ■ tank spawning, can deteriorate rapidly if eggs are Tank spawning not taken shortly after ovulation; ■ hand stripping, and they become “overripe” and can no Tank spawning is the simplest ■ surgically removing the eggs. longer be fertilized. In general, the method for obtaining a hatchery eggs of tropical and sub-tropical spawn. Brood fish of both sexes The method of choice depends on species of fish become overripe are placed together in the spawn- the fish species, hatchery facilities, more quickly than those that ing tank following injection(s). experience and skill of the hatch- spawn at cooler water tempera- Brood fish should not be disturbed ery staff, and the desired manipu- tures. The eggs of cold-water spe- and subdued lighting is recom- lations of eggs, sperm or fertilized cies remain viable for several days mended. The female ovulates eggs. after ovulation. Table 1 presents when she is physiologically ready. the reported maximum period be- The males stimulate the female to Taking the eggs tween ovulation and the deteriora-

Ovulation is the final phase of nor- mal egg development. The time Table 1. The maximum period between ovulation and deterioration of between the final or resolving the egg quality for various species of fish. dose of hormone and ovulation is referred to as the latency period. Bighead carp (Hypophthalmichthys nobilis) 50 to 80 minutes This is usually dependent on the species of fish, water temperature, Common carp (Cyprinus carpio) 50 to 80 minutes and hormone preparation used, It is especially important to know Grass carp (Ctenopharyngodon idella) 30 to 45 minutes the latency period when hand Rainbow trout (Salmo gairdneri) 7 days stripping or surgically removing the eggs. Check the literature for Red-tailed black shark (Labeo bicolor) 15 to 30 minutes the latency period for the fish species you are spawning. Snook (Centropomus sp.) 15 to 30 minutes Striped bass (Morone saxatilis) 15 to 30 minutes

* Institute of Food and Agricultural Sciences, Sturgeon (Acipenser sp.) 2 hours University of Florida White bass (Morone chrysops) 30 to 45 minutes release the eggs and fertilize the spawn. WATER INLET Better fertilization occurs if males are accustomed to the tank, and have been injected with a prepara- tory dose of hormone several days prior to and again at the same time as the female. Males can be used SPAWNING for several tank spawns, week TANK after week, until their milt flow di- minishes. Unless the males are aggressive toward each other, it is advisable to put two or three males for each female in a tank to ensure fertilization. If the spawning tank is of suffi- cient size, more than one female may be spawned in the same tank. Figure 1. Round spawning tank with screened egg collector. The presence of other individuals may help stimulate fish that are mass spawners. However, too many breeders in a small tank ■ might be disruptive to the spawn- Tank spawning has both advan- the surface area of substrate in ing process. tages and disadvantages. Advan- tanks is insufficient for large tages are: species that release large quanti- A round tank is advantageous for ties of sticky eggs. The-eggs species with non-sticky, floating or skill on the part of the hatchery clump together, resulting in fun- semi-buoyant eggs that spawn in a staff in predicting the exact time gus problems and poor hatch; river or estuary. The circular flow of ovulation or checking fe- and simulates the current in which males to verify ovulation is un- these fish naturally spawn. The necessary; ■ this method cannot be used if in- vigorous swimming action of the duction of polyploidy or other rapid deterioration of eggs in female in the swift water current is manipulations of eggs, sperm, the ovary after ovulation is not believed to assist in emptying the or fertilized eggs are a problem; ovaries. The eggs are carried with desired. the drain water from the spawning there is less potential of injury tank to a screened collector (Figure to the brood fish because it is Hand stripping 1). Eggs are then transferred to an unnecessary to check and strip incubator. the fish; and Hand stripping is commonly used for taking the spawn of many spe- Nest breeders and substrate less labor is required. cies of fish. Brood fish are sepa- spawners can also be tank rated by sex prior to hormone spawned if suitable nesting sites Disadvantages are: injection to prevent spawning in - - or spawning material are ■ the holding-tank. provided. When tank spawning a screened egg collector or suit- species that scatter sticky eggs, it is able spawning substrate is re- It is important to determine the advisable to place spawning mats quired; exact time of ovulation when hand stripping. However, the eggs of or brushes on the bottom of the ■ dirt and debris may be mixed tank. The eggs will attach to the cold-water species may remain vi- with the eggs, potentially caus- able for several days after ovula- substrate. Brood fish are removed ing problems during incubation; from the tank after spawning, un- tion; for example, in trout, eggs are less they provide parental care to ■ some females may not release usually stripped within 3 to 4 the eggs. Fertilized eggs are usu- all their eggs; days. The eggs of some species ally incubated in the spawning such as striped bass and white tank. ■ it is more difficult to accurately bass progressively clear or become estimate the number of eggs; transparent as they near ovulation. This visual cue is used by hatchery eggs until after the milt is added Surgically removing the workers to estimate the approxi- and mixed. Water and slime from eggs mate time of ovulation. An egg the vent and tail area of the female sample is taken by carefully insert- fish are dried with a towel. Water Because the internal anatomy of ing a tube (catheter) into the uro- activates the sperm and also fish varies greatly, hand stripping genital opening and examining the causes the opening through which may be difficult in some species. sample under a microscope (See the sperm enters the egg (micro- Sturgeon and paddlefish have no SRAC Publication No. 423, Deter- pyle) to close. For many fish, this ovarian sac; the eggs are released mining Sexual Maturity of Brood- closure takes place within only 45 into the abdominal cavity during stock for Induced Spawning of Fish). to 60 seconds. ovulation. The best method for tak- Eggs taken more than 15 hours being the spawn in many of these fore ovulation cannot be accu- To strip the eggs, the fish is held species is to surgically remove the rately staged using this method. slightly on her side, tail down; gen- eggs. The first indication of ovula- tle hand pressure is applied to the tion for sturgeon and paddlefish is For most species, ovulation can abdomen, moving toward the vent the appearance of several eggs best be verified by checking the fe- (Figure 2). The stream of eggs is stuck to the sides or bottom of the male to determine when eggs flow directed into a clean, dry bowl tank. The brood fish are usually freely from the vent. At least one positioned so that water from the left undisturbed for an additional hour prior to the earliest antici- fish does not drip onto the eggs. 1 to 2 hours, depending on the size pated time of spawning, female The head of small fish can be held of the female (small females 1 hour fish are captured and the process by one hand while the eggs are and large females 2 hours), to in- of checking to verify ovulation is stripped with the other. A cloth sure complete ovulation. initiated. Tropical species are usu- glove may be worn to help hold ally checked every 45 minutes the fish while stripping. Larger If the female sturgeon or paddle- until ovulation is verified, temper- species are either wrapped in a fish is to be saved, it is first anes- ate water species are usually towel and held by one or more thetized. The fish is temporarily checked every hour. It is not neces- hatchery workers while another placed in an aerated holding tank sary to take the fish out of the strips the eggs, or the fish maybe with MS-222. When opercular water to verify ovulation. The fish restrained on a padded table or movement slows and the fish is un- is turned belly up and gentle fin- stretcher for stripping. able to right herself when turned ger pressure is applied to the abdo- over, she-is then placed belly-up men starting at the pectoral fins, Good quality eggs usually flow on a stretcher. Two hoses are used moving slowly toward the vent. readily from the genital opening of to ventilate the gills during sur- Do not try to squeeze or force the the female and have little ovarian gery. One hose delivers aerated eggs from the fish; this will only in- fluid. If the ovarian fluid is wa- hatchery water, and the other de- jure the female. Frequent or rough tery or milky and many of the livers water from a recirculating handling of females retards ovula- eggs are cloudy white, this indi- tank containing aerated water tion, reduces spawning success cates poor quality eggs. with MS-222. During surgery, one and increases fish mortality. If only a few eggs flow from the vent when slight pressure is applied, partial ovulation has occurred; the fish should be released and checked again later. Attempt- ing to hand strip a female fish that has only partially ovulated will result in few mature eggs and physi- cal damage to the ovaries, preventing a complete spawn. When eggs flow freely from the vent, complete ovulation has occurred. The hatchery worker quickly plugs the flow of eggs by placing a thumb over the vent. Brood fish may be anesthetized with MS-222 for stripping, if necessary, after Figure 2. Hand striping the female by applying gentle hand pressure to the abdomen, moving toward the vent; the stream of eggs is directed ovulation is verified. into a bowl. It is important to insure that no water comes in contact with the

3 of the two hoses is always in the fish’s mouth, flushing the gills pro- viding dissolved oxygen. The hoses are exchanged so that the fish remains anesthetized but opercular movement continues. Too much anesthetic will kill the fish. Before surgery, an antibiotic solution is applied to the abdomen. An incision is made along the midline of the abdomen, and the eggs are carefully removed with a spoon (Figure 3). To avoid injur- ing the internal organs, only the eggs easily accessible are taken. The incision is closed with a half- circle surgical needle and suture Figure 4. Closing the incision with a half-circle surgical needle and suture material; a length of surgical tub- material. Note the surgical tubing, split lengthways, used to rein- ing, split length-ways, may be force the abdominal tissue. used to reinforce the abdominal tissue (Figure 4). The incision area is treated with an antibiotic before the fish is returned to the holding a bowl is placed under the vent, di- After fertilization, these eggs are tank. A high level of dissolved oxy- rectly below the incision. The eggs processed and incubated sepa- gen is crucial for rapid recovery of flow quickly from the abdomen, rately. the fish. pulled by the force of gravity. For delicate species that seldom A greater quantity of eggs can be Not all the eggs will be free in the survive the rigors of hand strip- obtained by sacrificing the female abdomen; some remain attached ping, humanely killing them and sturgeon or paddlefish. If the fe- to the folds of the ovaries. These surgically removing the eggs may male is to be sacrificed, it is: 1) eggs are removed by hand and be the best option. In addition, held in a net and killed with a placed in separate bowls. The proc- more eggs can usually be obtained blow to the head; 2) hung from a &s of ovulation will be completed by this method than by hand strip- hook; 3) the tail is cut off to bleed in the bowls. Within 15 to 20 ping. Once ovulation has been the fish, minimizing the contami- minutes these eggs can be fertil- verified (see section on “Hand nation of the eggs with blood; 4) ized. Visible amounts of blood, stripping”), the brood fish is held an incision is made in the abdo- which can inhibit fertilization, in a net and administered a blow men starting at the vent; and 5) may be present with the eggs. to the head to kill it. The eggs can then be removed from the fish by carefully cutting open the abdomen, pinching off the oviduct, and removing the ovaries individually. The total volume of eggs can then be gently squeezed out of the ovarian sac into a clean dry bowl to be fertilized. Hose Fertilizing the spawn Eggs are usually fertilized with fresh milt. Males are captured, wiped off, and held belly down over the bowl containing the eggs. Incision The portion of the abdomen poste- rior to the pelvic fins is gently mas- saged to extrude the milt onto the Figure 3. Surgically removing the eggs from the female sturgeon. Note the eggs. The number of sperm in a hose placed in the fish’s mouth to ventilate the gills. volume of milt is extremely vari- able, ranging from millions to bil- lions of sperm per milliliter.

4 Creamy-white milt contains more milt into the syringe. Care must dlefish, common carp, and chan- sperm per volume than grayish- be taken to insure that water, nel catfish become sticky after white milt. When available, milt urine, intestinal contents, slime, or coming in contact with water. from two or more males is used to blood is not mixed with the milt. During natural spawning, this insure fertilization of the spawn. It is best to collect and store milt stickiness causes the eggs to be- Individual males can be used to separately from each male to come attached to rocks, sticks, or fertilize more than one spawn. avoid contamination. aquatic plants. Catfish eggs are connected by a sticky matrix that The fresh milt is spread over the The milt is expelled into a sterile holds the eggs together in a mass eggs and thoroughly mixed by plastic bag, and antibiotic (e.g., 50 in the spawning cavern or con- hand, plastic spatula or feather. micrograms of dry streptomycin tainer. In the hatchery, this sticki- Only then is water added to acti- sulfate per milliliter of milt) may ness causes problems during vate the sperm. The sperm remain be added to control bacteria. The incubation. active in water for a very short bag is filled with oxygen, sealed time (less than 1 minute to 5 with a rubber band, and gently Silt-clay, bentonite and Fuller’s minutes), depending on the spe- swirled to mix the antibiotic. earth have been used to remove cies of fish and the temperature of Rough handling and shaking of the stickiness from the eggs of the water. Water also results in clo- milt may be detrimental to the many species of fish. Do not use sure of the micropyle of the egg in sperm; mixing should be done diatomaceous earth because the approximately the same amount slowly and gently. sharp edges of the diatoms will of time. Water is added to only damage the eggs. The dried mate- cover the eggs. Do not add too Bags of milt should be laid flat to rial is added to hatchery water much water because the sperm maximize the surface area of milt until a suspension is formed and a may be too diluted to ensure fertili- exposed to the oxygen and imme- residue accumulates on the bot- zation of the eggs. To fertilize diately stored on ice in a cooler or tom of the container. sticky eggs, see section on “Elimi- refrigerator. Do not freeze the milt nating the stickiness from eggs. ” as this will kill the sperm. Oxygen Paddlefish and sturgeon eggs are may be replaced and the milt commonly treated with silt-clay as The bowl containing the eggs and should be gently swirled in the soon as the first few sticky eggs sperm should be gently rocked, bag periodically to insure maxi- are noticed after fertilization, usu- swirled or stirred continuously for mum aeration. Milt that has been ally 1 to 4 minutes. The silt-clay several minutes to insure uniform contaminated with blood, slime, suspension is added to the fertil- distribution of the sperm and to etc., will appear to have congealed ized eggs at a ratio of 2 to 4 parts prevent the eggs from settling. Ad- and should be discarded. Milt has suspension to 1 part fertilized ditional water is added as the eggs been held in this manner for up to eggs. The mixture is gently stirred take up the water and enlarge three weeks; however, the quality by hand. Any clumps of eggs on (water harden). After several of the stored milt may deteriorate the side of the container are gently minutes, the fertilized eggs are with time. broken up. The suspension is transferred to the hatching appara- poured off the eggs, and fresh sus- tus. Fertilized eggs should not be The motility of stored sperm pension should be added every 10 exposed to direct sunlight. Sub- should be checked before it is used minutes to maintain proper tem- dued lighting should be used in to fertilize eggs (See SRAC Publica- perature and dissolved oxygen. the spawning and incubation area. tion No. 423, Determining Sexual Continue the process until the Maturity of Broodstock for Induced eggs do not stick to fingers or each Storage of milt Spawning of Fish). Milt with no or other when removed from the sus- low motility should be discarded. pension (minimum of 20 minutes). Milt can be collected from males When using stored milt to fertilize and stored up to three weeks prior the spawn, it should be mixed Urea and salt solution has been to stripping eggs. Males are cap- with water first and then gently used to remove the stickiness from tured and anesthetized, if neces- shaken for five seconds before common carp eggs. The addition sary. The fish is then turned belly being added to the bowl with the of water to common carp eggs up, and the vent area dried by blot- eggs. Remember, the sperm re- and milt will result in their stick- ting with a towel. The area just be- main active in water for a very ing together in a clump within a hind the pelvic fins is gently mas- short period of time, so this must few seconds. By using urea-salt saged toward the vent to extrude be done quickly. solution instead of water, the the milt. The first few drops of spawn can be fertilized without milt are wiped away. The milt is Eliminating the stickiness the eggs sticking. A commonly collected by inserting a plastic from eggs used solution is prepared by dis- tube attached to a syringe into the solving 30 grams of urea and 40 urogenital opening. Suction is ap- Ovulated eggs of many species grams of salt in 10 liters of hatch- plied while stripping to draw the such as white bass, sturgeon, pad- ery water. The solution is added

5 to the eggs and sperm. The initial minutes to eliminate any remain- bators. The solution is prepared by volume of solution added is ap- ing stickiness. The water inlet mixing 15 grams of sodium sulfite proximately 25 percent of the vol- valve to the jar is opened, flushing with 1 liter of hatchery water. If ume of eggs. The mixture is the tannic acid. the water supply has low alkalin- gently stirred continuously with a ity, the pH must be adjusted back feather, plastic spatula, or by Tannic acid alone has also been to that of the hatchery water sup- hand. It has been observed that used to remove the stickiness from ply using 10 percent hydrochloric the motility of common carp white bass, sturgeon, and paddle- acid (HCl). The egg mass is re- sperm lasts much longer in the fish eggs. A tannic acid solution moved from the spawning con- urea-salt solution (20 to 25 of 150 milligrams/liter is often tainer at least 24 hours after minutes) than in water (1 to 2 used for this purpose. This solu- spawning and placed in a plastic minutes). As the eggs water hard- tion is prepared by adding 0.75 pan. One liter of the sodium sulfite en, additional solution is added. grams of tannic acid to 5 liters of solution is added per 500 grams of A portion of the solution with the hatchery water in a McDonald jar eggs. The egg mass is gently dissolved sticky material is poured just prior to adding the eggs. The kneaded and stirred until the ge- off at intervals and replaced. After solution is aerated with a latinous matrix is completely dis- about 1 to 1.5 hours the water weighted air stone at the bottom solved. The entire contents of the hardening process is completed. of the jar. The fertilized eggs (1 pan is poured into a hatching jar The eggs are then transferred to a minute after the water is added to and the water flow is adjusted to tannic acid solution (750 mg/L) activate the sperm) are placed in gently tumble the eggs without for 5 seconds to eliminate any re- the jar. Aeration is adjusted to just washing them from the jar. No maining stickiness. To remove the keep the eggs in suspension. The more than 1400 grams of eggs tannic acid, the eggs are thor- air stone is removed after 10 to 12 should be incubated in a seven- oughly rinsed with fresh water. minutes, and the water inlet valve liter hatching jar. Dead eggs, white is opened to the jar. Although not in color, float near the top of the The urea and salt solution has also absolutely necessary, an excess egg mass and should be removed been used to remove the stickiness quantity of milt appears to help re- by siphon to prevent fungus prob- from white bass eggs for the pro- duce stickiness of the eggs. lems. duction of hybrid striped bass. A solution is prepared in a McDon- When the alkalinity of the water is ald jar with 5 liters of hatchery above 200 milligrams/ liter, addi- Conclusions tional tannic acid maybe needed. water, 15 grams of urea and 20 The spawn can be taken from fish grams of salt. The solution is aer- However, an excessive amount of tannic acid can strengthen the egg by several different methods, in- ated with a weighted air stone at cluding allowing the fish to spawn the bottom of the jar until the shell, resulting in difficulties at hatch. For white bass eggs, an ad- in the tank, hand stripping, and chemicals are dissolved. A small surgically removing the eggs. amount of the solution is added to ditional disadvantage of this proce- dure over the use of urea-salt is When hand stripping or surgically cover the eggs and sperm, fertiliz- removing the eggs, either fresh or ing the spawn. The mixture is that some batches of eggs are extremely sticky, especially if only a stored milt can be used to fertilize gently stirred. Four minutes after the spawn. Ovulated eggs of the solution is added to the egg- limited quantity of milt is available. In addition, the resulting egg many species of fish have a sticky sperm mixture, 400 milliliters or exterior. Several common prepara- less of fertilized eggs are placed in shell is opaque rather than clear, preventing microscopic examina- tions can be used to eliminate the each jar with the solution. Air sticky layer of fish eggs. flow is adjusted to keep the eggs tion of the developing embryo. in suspension without rupturing A sodium sulfite (Na SO ) solu- Induced hatchery spawning re- them. After 6 minutes, the urea 2 3 tion can be used to dissolve the quires a continuous series of steps. and salt solution is poured off the gelatinous matrix of catfish egg A mistake during any phase of the eggs, and 0.75 grams of tannic acid masses so they may be incubated process can diminish or com- mixed in 5 liters of hatchery water in hatching jars, eliminating many pletely obliterate the success of the (150 milligrams/liter) is added problems associated with tradi- project. Consistent performance and aerated for an additional 6 tional paddle-wheel-trough incu - requires strict attention to detail.

The work reported in this publication was supported in part by the Southern Regional Aquaculture Center through Grant No. 89-38500-4516 from the United States Department of Agriculture.