Winter Flounder Essay, Research Paper

PROCEDURE

Between January and March 1996, sexually mature winter flounder were captured from Shark River, NJ by Steve Teluvech of Richard Stockton College Nacote Creek Marine Science Research Center. The flounder were then kept in an aerated tank with a maintained temperature between 1-10(C and a salinity between 11.4 – thirty-three ppt in the said research center.

Each individual flounder was injected with a prepared carp pituitary hormone extract (GtH) according to their body weights to induce spawning. Using a 20 microliter syringe, the extract was injected into the intraperitoneal cavity. Two injections, within four hours, per specimen were given per day. After the appearance of a swollen abdomen, the injections were terminated, The flounder were allowed time for ovulation before the milt (semen) and the eggs were stripped and collected into a container.

The eggs and the milt were allowed to mix for five minutes to permit optimal fertilization. Then, the egg mixture was washed with clean sea water, with a 20-30 ppt salinity and 5-10(C temperature, to remove excess organic matter. The mixture was bathed in a 1% bentonite solution, rinsed and then equally divided and placed into aerated 2 liter plastic containers. To maintain the temperature, the containers were then placed in the same tank with the flounders. Aeration was controlled to maintain adequate dissolved O2 level. A certain volume if fungicide was added to prevent fungal growth.

Observations of the development of the eggs were taken daily under dissecting microscopes. Unfertilized eggs were collected and discarded. A sample of eggs in different stages of development was also collected and preserved in 10% formalin. Twenty percent of the sea water of the containers was also a changed daily and transferred into another container.

INTRODUCTION

Winter flounder (Psuedopleuronectus Americanus) is a benthic marine fish that occurs from the Gulf of St. Lawrence to the Chesapeake Bay. They do not spawn well in laboratories or aqua cultured systems. Due to decreasing natural stocks from over fishing, Aquaculturists have tried successfully to spawn winter flounder. Current spawning methods require an injection of every specimen that becomes time consuming and stressful. The goal of this project was artificially to induce spawning by injecting carp pituitary hormone extracts. Hatchlings will then be raised eventually to find alternative ways of spawning winter flounder.

In this research project carp pituitary extract Gonadotropin hormone (GtH) will be used to induce winter flounder spawning. GtH induces ovulation and eventual spawning. Once spawning occurs fertilization will be accomplished artificially and eggs will be incubated for the duration of development. The eggs will be kept under the following conditions: temperature between 5-10(C and salinity between 25-35 ppt. Hatchlings are expected in the second and third weeks following fertilization. After hatching, cultured artemia (brine shrimp) will be introduced to encourage feeding and growth.

ABSTRACT

Winter flounder (Pseudopleuronectes americanus) were obtained from Shark River jointly by the NJ Department of Environmental Protection and Nacote Creek Marine Station of Stockton College between the months of January and March. They were kept in a refrigerated aquarium at Nacote Creek Marine Science Laboratory. The flounder were injected with carp pituitary extract that contained gonadotropin hormone (GtH), at a dosage of 0.5 mg acetone dried powder per Kg flounder body weight. This hormone initiated oocyte development, ovulation and eventual spawning. Three of the four females and all males injected spawned and produced milt, respectively.

Spawning females were stripped by hand and about 674,000 eggs were obtained in total from two individuals. The eggs were fertilized and were treated with 1% bentonite solution to remove the egg jelly coating. They were partitioned into six two-liter plastic containers. The eggs were aerated and about 25% of the water in each container was changed each day. Temperature in the tank was maintained between 7-10(C and salinity between 25-30 ppt. Development of the fertilized eggs was monitored with a dissecting microscope. Dead or unfertilized eggs were discarded. About 1,000 hatchlings were separated from the eggs. A hatchling success rate of about 0.15% was realized. After yolk sac absorption, artemia was introduced for feeding.

The above experience will be used in developing a stock of transgenic winter flounder using carp gonadotropin DNA to spawn them on demand and out of a season.

RESULTS

The following observations were made after the administration of a carp pituitary hormone (GtH) to the winter flounder. First, two injections, within four hours, injected per day were sufficient in artificially inducing hydration and spawning. Second, a period of ten days was needed for ovulation of the flounder and optimal development of the gametes. Finally, eggs and milt collected after the rest period were more likely to be fertilized compared with samples collected before the rest period.

The stripped females collectively produced about 673,700 eggs. Using a dissecting microscope, the yolk, perivitelline space, germinal vesicle, and micropyle were observed. This data suggested that the eggs were suitable for fertilization.

The dry method of artificial fertilization was more effective than the wet method. About 80% of the total numbers of eggs fertilized using the dry method displayed further development, while eggs fertilized using the wet method fertilization did not significantly show any further development.

The following observations were made after fertilization. After two hours the first division was present in most eggs. The morula stage, a cap-like formation of cells on top of the yolk, was observed after 23 hours. The stage observed after 24 hours was characterized by further cell division and the egg cap was extended over part of the yolk.

Four days after fertilization the neural tube and developing cephalic region and optic anlagen was observed in some eggs. The embryo was also transparent. These stages were observed through the eighth day.

Myomeres and eye cups were observed after eight days. The embryos had grown to a length greater than the egg diameter and were folded around the inside the yolk. Hatchlings were observed after nine days. All hatchlings had a yolk sac larger than the diameter of their body. Larger hatchlings displayed a darker band of chromatophores toward the posterior end of the body on the tail region. Many hatchlings exhibited phototaxis.

On the twelfth day, use of the yolk contributing to growth caused reduction of yolk size. By the fifteenth day of development about 940 hatchlings were collected and placed into a separate 2 liter bottles. At this stage larvae had pectoral fins, and well-developed eyes. They reached a size of 4-6 mm.

DISCUSSION AND CONCLUSION

The GtH hormone used on winter flounder was successful in inducing spawning. All of the injected flounder had a reaction to the GtH hormone, except one female that did not spawn. This could have been a result of premature stripping efforts.

The development and hatching of the eggs were greatly accelerated as compared with reported data obtained by related research. This could be explained by the results found by Carolyn A. Rogers of NOAA corresponding to temperature and duration of hatching intervals.

Most of the hatchlings were incubated in one 2-liter plastic bottle. Eggs incubated in the 2-liter bottles had a better hatching success rate than those in plastic trays with surface aeration. The bottle that was most successful in production of hatchlings was kept at a lower aeration level than other bottles. The inference could be drawn that less dissolved oxygen is needed by the eggs and that they should have little agitation during incubation.

In conclusion, the GtH hormone and the experimental protocol were successful in artificial spawning and rearing of winter flounder. To get optimal results, the following conditions are suggested:

Aeration must be controlled to maintain a constant oxygen level in the water and to reduce agitation to the developing eggs.

Temperature must be maintained within a range of 1-6(C, for optimal egg development.

Salinity should be maintained within 20 to 30 ppt.

Dry method of fertilization is recommended.