Determining Sexual Maturity of Broodstock for Induced Spawning of Fish

SRAC Publication No. 423

Southern Regional Aquaculture Center

November 1991

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

Hormones injected for induced Sampling eggs and sperm with a microscope (e.g., 60x). The spawning of fish do not produce male is turned belly up, and the eggs and sperm (gametes); they Sampling the eggs and sperm of vent area is dried by blotting with only trigger the release of fully de- the brood fish eliminates the guess- a towel. The area just behind the veloped gametes. Fish must not work in determining the stage of pelvic fins is gently massaged to- only be sexually mature but also in sexual development. Brood fish ward the vent to strip the milt. The the advanced stage of sexual devel- must be sampled quickly and care- first few drops of milt are wiped opment before induced spawning fully to minimize physical injury away. A sample of milt is col- will be successful. and stress. The importance of lected by inserting the tip of a eye- proper handling cannot be overem- dropper into the urogenital The external appearance of brood phasized. Before sampling the opening. Suction is applied while fish has long been used to assess testes or ovaries, the fish maybe stripping to draw milt into the eye- the stage of sexual development. quieted, if necessary, with anaest- dropper. Care must be taken to in- In some species, males change in hetic such as MS-222. It is best to sure that water, urine, intestinal appearance during the spawning keep the fish in the water when contents, slime, or blood are not season (e.g., vivid breeding colors sampling. Time spent with the fish mixed with the milt. A drop of in cichlids, hooked jaw in salmon, out of the water can mean the dif- milt is placed on a cover slip, and and roughened texture of the pec- ference between a perfect spawn, a drop of water is placed on a toral fins or head of cyprinids). no spawn, or death of the fish. glass slide. The cover slip with the These physical changes make it re- Sampling the testes milt is placed on the drop of latively easy to identify sexually water, gently pressed over the mature males. However, secon- Milt can usually be stripped from slide, and immediately observed dary female sex characteristics males of most species when they under the microscope. The sperm such as plumpness of the abdo- are ready for spawning by apply- remain active in water for a very men and redness of the vent are ex- ing gentle pressure to the abdo- short period of time, usually less tremely subjective and can be men between the pelvic fins and than 1 to 5 minutes, depending on misleading. the vent. The color of the milt is the species of fish and the tempera- Taking brood fish from the spawn- usually creamy-white to grayish- ture of the water. Males with milt ing grounds at the height of the white. Number of sperm in a vol- that have no or low motility spawning season eliminates most ume of milt is extremely variable, should be discarded. of the guesswork. However, the ranging from millions to billions Sampling the ovaries capture of broodstock from non- of sperm per milliliter. Creamy- spawning areas or use of hatchery white milt contains more sperm Several tests are available to deter- reared fish may be preferable or per volume than grayish-white mine the developmental stage of the only option available. milt. the eggs in the fish’s ovary. Two common methods are: 1) egg ap- * Institute of Food and Agricultural Services, Sperm viability usually can be de- University of Florida termined by observing motility pearance; and 2) physiological state of the egg. Both require that an egg sample be taken from the fish. For species that reproduce during a precise spawning season, only a small number of females need be sampled to get an indica- tion of their stage of development. However, if there is a wide vari- ability in egg development, it is best to sample each female. The ovaries can be sampled with either a rigid or flexible tube (catheter). Rigid catheters are usually made from lengths of glass or hard plastic tubing. Flexible cathe- Figure 1. Collect an egg sample by inserting a catheter through the genital ters are prepared from lengths of opening, into the ovary. polyethylene or vinyl tubing. The catheters must have an outer diameter small enough to be inserted tube. The incision is closed with a through the genital opening and half-circle surgical needle and su- sufficient inner diameter to accom- ture material (Figure 3); the area is modate the eggs. The leading edge then treated with an antibiotic. of the catheter should also be Cavity smoothed or rounded to prevent Determining egg maturity damage to the fish. Figure 2. A metal or plastic rod Visual examination To collect an egg sample, the cathe- with a rounded-conical ter is inserted through the genital end and a cavity cut in The diameter and general appear- opening and rotated, while gently the rod used to sample ance of the egg are indicators of de- threading it down the oviduct into the ovaries of Chinese velopment. Approximate the ovary (Figure 1). Forceful pres- carps. diameters of ripe eggs of different sure will puncture the oviduct or species of fish are presented in ovarian wall, Sampling with a along the belly of the fish. First, a Table 1. The color of ripe eggs flexible catheter minimizes dam- small amount of physiological sa- also varies with the species of fish. age to the oviduct, and it will not line solution is drawn into a flex- Immature eggs are much smaller break off in the fish if she struggles ible tube. The tube is inserted than ripe eggs and are usually when the tube is inserted. If resis- through the incision into the nearly clear or opaque white or tance is felt, the tube should be re- ovary, and the saline solution is re- yellow, depending on the fish spe- moved and reintroduced at a leased. Suction is applied to draw cies. Eggs that have begun to slightly different angle. If neces- a small number of eggs into the break down (resorb) in the ovary sary, suction may be applied to the appear whitish in color. Under catheter by mouth or a syringe to draw a small number of eggs into the tube. In China, a metal or plastic rod Suture with a rounded-conical end and a cavity cut in the rod (Figure 2) is used to sample the ovaries of Chi- nese carps. The rounded tip is inserted in the genital opening, and because of its shape, does not puncture the curved oviduct. Once the cavity of the rod is in the ovary, it is rotated one full turn and withdrawn. The eggs are re- Surgical needle tained in the cavity. An egg sample can also be taken Figure 3. Use of a half-circle surgical needle and suture material to close the from sturgeon and paddlefish by incision used to sample the ovaries of paddlefish and sturgeon. making a small (20 mm) incision Table 1. Approximate diameter of mature eggs for various species of fish. Species Diameter Bighead carp (Hypophthalmichthys nobilis) 0.9-1.2 mm Channel catfish (Ictalurus punctatus) 2.3-2.8 mm Common carp (Cyprinus carpio) 0.9-1.2 mm Grass carp (Ctenopharyngodon idella) 0.9-1.2 mm Gray mullet (Mugil cephalus) 0.6-0.8 mm Figure 6. Appearance of mature Red-tailed black shark (Labeo bicolor) 1.0-1.4 mm egg under the microscope. Note that the Snook (Centropomus sp.) 0.6-0.7 mm nucleus is near one Striped bass (Morone saxatilis) 1.0-1.2 mm edge of the egg. Sturgeon (Acipenser sp.) 3.5-4.0 mm White bass (Morone chrysops) 0.6-0.7 mm hormone-induced spawning. The time interval for migration of the nucleus of the egg varies between the microscope, eggs that have using this method. Immature eggs species and is affected by environ- begun to resorb appear irregular appear much smaller and remain mental parameters, especially in composition, and the egg con- opaque following hormone injec- water temperature. tents appear to have pulled away tion. The nucleus can be observed in from the cell membrane (Figure 4). Eggs that have begun to resorb Position of the nucleus in the some species (e.g., goldfish, common carp, grass carp, bighead often appear soft or partially de- egg carp, snook, red drum, pompano, flated rather than firm and round. Movement of the nucleus (germi- red-tailed shark, rainbow shark) nal vesicle) from the center of the by placing a cover slip over the egg to the edge (germinal vesicle eggs on a glass slide. The weight migration) is a preliminary step to of the cover slip slightly com- ovulation. Observing the position presses the egg. When lighted of the nucleus is a good method of from below, the nucleus appears determining egg development. as an translucent circle in the The nucleus of an egg in the rest- opaque egg when viewed under a ing phase is located in the center microscope or hand lens. The ori- (Figure 5). As the egg matures the entation of the egg on the slide nucleus moves to the end (animal will determine the observed posi- pole) that contains the opening(s) tion of the nucleus (i.e., if the ani- Figure 4. Under the microscope, (micropyle) through which the mal pole is toward the cover slip irregular appearance in sperm enters. When the nucleus is or slide, the nucleus will appear to composition of eggs near one edge of the egg (Figure be in the center). However, if the that have begun to re- 6), the eggs are considered ripe nuclei of a great majority of the sorb. Note that the egg and the fish should be injected for contents appear to have eggs in the sample are near the pulled away from the edge or off-center, there is a high membrane. probability that the eggs are ripe. Clearing solutions have been used The eggs of some species of fish to facilitate viewing of the nucleus, (e.g. striped bass, white bass, especially in species with eggs that snook) progressively clear or be- are too opaque to use the “cover come transparent as they near ovu- slip” technique. A solution of 60 lation. This process is the result of percent ethanol, 30 percent forma- coalescence of the oil droplets into lin, and 10 percent glacial acetic several large droplets and then acid by volume has shown to be into a single oil globule. This clear- Figure 5. Appearance of an egg effective. Within 5 to 20 minutes ing of the egg is used to estimate under the microscope in the solution, the nucleus of the the time of ovulation. Only eggs in the resting phase, with egg can be seen. the nucleus located in of fish that are within 15 hours of the center. To determine the position of the ovulation can be accurately staged nucleus in sturgeon and paddlefish eggs, the eggs are boiled in (germinal vesicle breakdown) The nucleus is observed in the egg water for 5 to 8 minutes and then occur just prior to ovulation as a using the procedures outlined in cooled. A razor blade is used to result of the first meiotic division. the section titled “Visual exami- cut the egg in half along its length, Eggs in the advanced stage of de- nation.” If no nucleus can be de- bisecting the animal pole. The velopment will undergo germinal tected (germinal vesicle break- position of the nucleus can then be vesicle breakdown and sometimes down) in a high percentage (80 to determined using a hand lens or ovulation outside the body (in 100 percent) of the eggs, the fe- microscope. The nucleus appears vitro) when placed in a solution males are considered to be suitable as a dark sphere (Figure 7). containing steroids or gonadotro- for hormone-induced spawning. pin. This may be used as a test to determine if egg development is Conclusions sufficiently advanced to respond External appearance of brood fish successfully to injected hormones. has long been used to assess the The eggs are incubated in a solu- stage of sexual maturity. In some tion containing a maturation-in- species of fish, the males change in ducing substance (e.g., progeste- appearance during the spawning rone). Progesterone (4-Pregnone- season. Milt can usually be 3,20-dione) is insoluble in water stripped from males of most spe- and must first be dissolved in cies when they are ready for alcohol. An effective solution for spawning. However, secondary Figure 7. Appearance of mature hormone assay is prepared by mix- female sex characteristics are ex- sturgeon and paddle- tremely subjective and can be mis- fish egg under the micro- ing 10 mg of progesterone in 10 ml scope. Note that the of 100 percent ethyl alcohol; the leading. Several methods are nucleus is near the final concentration is 1.0 mg/ml. available to determine the stage of animal pole of the egg. Then 0.2 ml of the progesterone so- development of the eggs in the lution is added to 20.0 ml of tissue fish’s ovary. These require that a culture media (e.g., L-15 Medium sample of eggs be taken from the Unfortunately, techniques have Leibovitz, physiological saline) in fish. The diameter and appear- yet to be developed to observe the a sterile disposable petri dish and ance of the egg and the position of position of the nuclei in the eggs of gently swirled to mix. The eggs the nucleus in the egg are visual in- all species of fish (e.g., catfish). are added to the petri dish with dicators of development. The ster- The diameter and general appear- the solution and are allowed to in- oid assay procedure determines ance of the egg are the best indica- cubate at the spawning tempera- the physiological response of the tors of development for these ture of the species for at least 12 eggs to hormones. An under- species. hours. The progesterone artifi- standing of sperm viability and cially stimulates egg maturation. egg stage development will Hormone assay greatly improve the success of hor- At the end of the incubation pe- mone-induced spawning of fish. Changes during final egg matura- riod, the eggs are examined for the tion such as breakdown of the nu- presence or absence of the nucleus. clear membrane of the egg

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.