D.E. Jory-1996
Aquaculture Magazine, 22 (6): 77-86
This second column on shrimp hatcheries will be a brief over view of broodstock maturation and its relevance to shrimp farming. Maturation of wild-caught penaeid broodstock is routinely done in many areas, but maturation of captive broodstock over several generations and the "closing" of the life cycle has only been achieved on a consistent, dependable basis for a handful of penaeid species. Shrimp hatcheries around the world continue to be heavily dependent on wild broodstock.
The importance of maturation is obvious, as the ability to produce seedstock on demand, consistently and in sufficient numbers to support the industry must be a major objective to maintain shrimp farming as a strong, environmentally friendly and viable industry. The importance of domestication and stock selection for marine shrimp culture has already been emphasized in previous contributions...
Various approaches are used to spawn penaeid shrimp. One way is to capture or procure from suppliers wild, sperm-bearing females that can spawn right away; this strategy normally produces high quality eggs and larvae. The second way is to obtain wild adults and mature and spawn them. The third approach is to mature and spawn adult shrimp that have been reared in captivity. The first two approaches are widely practiced around the world in most shrimp farming countries. At least 26 penaeid shrimp species have been matured and spawned in captivity to produce viable eggs at experimental and pilot scales. However, only a few species are routinely matured and spawned in captivity on a commercial (consistent, large-scale) basis, and just a handful have been bred over several generations in captivity. The first two spawning strategies mentioned above imply the continuing dependence of hatcheries on wild stocks and an inherent failure risk due to the known presence of various viruses in, and the variable availability of, wild shrimp stocks. The third strategy implemented by rearing succeeding generations in captivity is the only one that will lead to successful stock domestication, selection for commercially important traits like fast growth and disease resistance, and eventually to stock improvement.
Some penaeid species appear more suited than others for captive maturation. The giant tiger shrimp (P. monodon) is the most important farmed shrimp species and has dominated world production for many years, but it is comparatively more difficult to mature in captivity than other species. Giant tigers comprised 60% of all farmed shrimp in 1995 (around 500,000 metric tons); this production was almost entirely based on wild-caught spawners. It was recently estimated that over one million wild broodstock of giant tiger shrimp must be captured each year to provide needed seedstock and sustain the southeast Asia shrimp farming industry. Other important penaeid species - such as P. vannamei and P. stylirostris - are relatively easier to mature in captivity.
Design of Maturation Facilities
Maturation facilities should be located on sites with nearby access to warm water with oceanic characteristics; otherwise, an elaborate water supply/filtration/conditioning system may be required to guarantee the water quality needed. Large hatcheries typically have infrastructure to carry out broodstock maturation, although there are backyard, family operated small scale maturation facilities in many shrimp farming countries.
The maturation section of a large hatchery is normally a functional area that is isolated from other sections, to maintain noise level and stress caused by human activity at a minimum. Maturation tanks are usually arranged in batteries or rows in a room designated for this purpose. The typical tank used is round, about 3-5 m in diameter and 60-100 cm in height, and gently sloped towards the center drain to facilitate removal and siphoning of uneaten food and other undesirable debris. The preferred color used on the inside of maturation tank walls is black, which reduces the occurrence of animals hurting themselves by swimming or jumping against the tank walls. Tanks with white bottoms make it easier to source species with darker shells. Vinyl or PVC liners are commonly used because of the reduced construction costs, injuries to animals caused by jumping into tank walls are diminished, and tanks with liners are easier to clean and disinfect. For burrowing penaeid species a substrate (sand or pebbles) is sometimes provided, but in general substrates are not used in maturation tanks due to the increased management input needed and the increased potential for buildup of organic residues, and also to expedite daily tank cleaning procedures that can be stressful to maturing animals. Maturation areas are generally kept off limits to most hatchery visitors. These areas must be quiet and movement around and in tanks kept to a minimum, because animals are easily disturbed and stressed, with adverse effects.
In examining the environmental characteristics of penaeid shrimp spawning grounds, it is evident that conditions are relatively stable, and this is emphasized in the design and operation of maturation facilities. There are a number of environmental parameters that are amenable to manipulation and are critical for successful maturation of captive broodstock. Each species has an optimum value within a generally narrow, common range for these various parameters, which include water temperature and salinity, photoperiod and light intensity, lunar phase, maturation room and tank characteristics, and diet. Both the value of these parameters and their rate of variation over time are very important to stimulate the onset of the reproductive process. Among the critical requirements for successful maturation of captive stocks it is important to optimize environmental conditions by duplicating or intensifying those factors known to stimulate reproduction, and also to maintain environmental conditions as stably as possible.
The water supply system should be designed to continually provide maturation tanks with clear, unpolluted water with oceanic characteristics, and a daily exchange capacity of 200% to 300%. For most penaeid species the optimum water temperature for maturation is 27 to 29 C; each species has its own minimum and maximum temperature values depending on the source one consults. In general, the temperature should be maintained stable and preferably not vary more than 1-2 C over 24 hour periods. An oceanic salinity of 30 ppt is considered optimum, although maturation may occur between 28 and 36 ppt, and pH values should be maintained between 8.0 and 8.2 . The addition of buffers, salt and trace metals are normally not recommended but are sometimes necessary.
Penaeid shrimp are separated into two groups based on their thelycum structure. The thelycum is an external structural component of the penaeid female reproductive system, and consists of anterior and lateral plates located on the ventral region of the female. The open-thelycum or "white" shrimp are five species indigenous to the western Hemisphere (the Pacific Ocean species P. vannamei, P. stylirostris and P. occidentalis, and the Atlantic Ocean species P. schmitti, and P. setiferus), and the closed-thelycum or "brown" shrimp species (notably P. monodon, P. japonicus, P. merguiensis, P. indicus and several others). Penaeid shrimp have a protective exoskeleton or shell which is shed or molted periodically as a normal event in the life cycle of the animal. Open thelycum species molt, mature, then mate and spawn, while closed thelycum species molt, mate and then mature and spawn. Maturation management procedures are somewhat different for open- and closed-thelycum species. For example, natural photoperiod is normally used in maturation facilities for most closed thelycum species, while for open thelycum species a reversed photoperiod regime is commonly established (using artificial lights) so that animals will spawn during the normal, daylight working hours of staff.
Maturation Procedures
The maturation process is not a complicated one. It basically consists of selecting or sourcing good prospective broodstock (screened for viruses) and holding the animals under the best possible conditions, including stable, optimal environmental conditions, minimal stress and adequate nutrition using both natural and artificial diets. Successful maturation requires selection of an adequate site, well designed maturation infrastructure (tanks and water supply system) and competent management procedures. It is very important to exclude and control opportunistic shrimp pathogens such as various bacteria, fungi and protozoa. This is accomplished by maintaining the best water quality possible and by periodic prophylactic treatments using various chemicals. In addition, female shrimp usually undergo a procedure called unilateral eyestalk ablation to stimulate ovarian development. Water temperature and photoperiod in the holding tanks are routinely manipulated to simulate optimal conditions and any changes the animals would normally experience in their natural environment.
It is critical for successful maturation to start with the best possible broodstock animals. Wild mature females are widely preferred as broodstock, but pond reared broodstock are also used extensively, particularly in areas with limited seasonal availability of wild broodstock or where the species cultured is not endemic. Examples are ongoing breeding programs in the U.S. (U.S. Marine Shrimp Farming Program and others), in Tahiti and New Caledonia (AQUACOP), Venezuela and other countries where successful rearing of exotic shrimp species through several generations in captivity has been accomplished.
Maturation tanks are stocked at densities of 3-8 animals/m2 depending on shrimp size, and at ratios from 1:1 to 1:4 male to female for closed thelycum species, and 1:1 to 3:1 male to female for open thelycum species. Unisex tank systems where females, once mature, are transferred to all male tanks, have been tested with results comparable to those obtained in conventional tanks.
Eyestalk ablation is a procedure widely used to induce maturation in penaeid shrimp. Since the 1940's it has been known that eyestalk ablation had a stimulating effect on decapod crustacean reproduction, but the technique was not incorporated into shrimp culture until the 1970's. In early attempts ablation was done bilaterally (both eyestalks removed) and resulted in high mortality rates. Unilateral eyestalk ablation reduced mortalities to acceptable levels while stimulating maturation. Normally only females are ablated. The procedure has been tested in males of several penaeid species with varying results, but it is rarely done on males at commercial level maturation facilities, as it is not considered necessary and may in fact be detrimental by causing precocious maturation. Female shrimp have a structure called the "X" organ in their eyestalks which produces a hormone that inhibits ovarian maturation. Under natural environmental conditions the levels of this hormone sharply decrease in animals during their migration to their spawning grounds, very likely an evolutionary response to environmental clues or stimuli. It is possible to mature, mate and spawn several shrimp species without resorting to eyestalk ablation, by subjecting animals to a controlled, manipulated photoperiod and water temperature regime. However, this has only been successful at experimental scale levels and has yet to be proven on a continuous, on demand commercial scale system.
Eyestalk ablation is a relatively simple procedure and is normally done only on shrimp that are hard-shelled, not on animals that are about to molt or that have recently molted. Prospective broodstock are normally acclimated for several weeks in maturation tanks prior to undergoing ablation. There are various procedures to perform ablation: severing the eyestalk with scissors; enucleation by pinching, crushing and squeezing the eyeball and its contents after an incision on the eyeball has been made; by tight ligation of the eyestalk (the eye falls out in a few days); and by cauterizing the eyestalk and eyeball with an electrical instrument or using silver nitrate. All methods are effective in removing the X organ, but the enucleation procedure is considered the simplest and easiest to perform, as it requires only one person and the wound heals fast and without antibiotics. To reduce stress and minimize mortality the animals are immersed in cold water before and after the ablation procedure is performed. The timing of ablation is very important, and the results can vary depending on the season and stage in the molt cycle. The best results are obtained when ablation is synchronized with the molt cycle and performed as animals are entering their reproductive peak. Ablation has been reported to have adverse physiological consequences in some closed-thelycum species, and recent and current research efforts are aimed at the possibility of using other approaches to stimulate ovarian maturation, including various hormones and same-species ganglion implants.
Proper nutrition is another factor critical for the success of maturation programs, as a proper diet promotes sexual maturation and mating, and improves fertility and offspring quality. Maturation diets typically include combinations of natural food organisms and commercial, dry pelleted feed supplements. Natural organisms commonly used include various mollusks, crustaceans, fishes and bloodworms. The most common animals used are squid and bloodworms. Bloodworms are marine polychaetes considered by many hatchery managers to be an essential component of the maturation diet, and although expensive they are used extensively in maturation facilities around the world. These polychaetes are rich in the polyunsaturated, long chain fatty acids (PUFAs) that are essential for shrimp to mature. High demand for bloodworms has resulted in strict export regulations being imposed in several countries that harvest these polychaetes.
Five ovarian maturation stages are recognized in female penaeids. Immature animals are stage I; early maturing animals are stage II; late maturing animals are stage III; stage IV is ripe or mature, and stage V is a spent ovary after spawning. The progression in ovary development can be assessed by holding animals against a light source (or by shining a flashlight from above while animals remain in the tanks) to determine ovary size and coloration. In a stage I animal the ovaries are very thin and practically invisible strands. As the ovaries develop through stages II to IV they can be seen becoming progressively thicker and darker through the animal's exoskeleton. Visual assessment is harder on species with a darker shell. Mating in penaeid shrimp is characterized by particular courtship behavior, and venous pheromones are involved in attraction. Different behavior and rituals involving protracted, elaborate chasing, contortions and other activities have been observed and described in detail for different species. Mating in open thelycum penaeids normally occurs only late in the day between males and females that have hardened exoskeletons, with males attaching a complex sperm packet or spermatophore on the ventral surface of the female's body and close to the gonopores. Sperm extrudes from the sperm packet and fertilization occurs as the female spawns and discharges its eggs and these come into contact with sperm in the attached packet. Mating in closed thelycum shrimp usually takes place during the night. A male with hardened shell will mate with a female that has just molted (and is therefore still "soft") by inserting its sperm packet into the seminal receptacle, an opening in the female's shell in the same location where the spermatophore is attached in open thelycum shrimp. The female carries the sperm packet internally until it spawns or molts, and mating typically occurs again shortly.
Penaeid shrimp are very fecund animals, producing between 100,000 to 1,000,000 eggs per spawning in the wild, and about 50,000 to 400,000 eggs per spawning in captivity. The species and size of the spawner determines spawn size, with larger species and larger animals producing more eggs per spawn. Ablated females will normally spawn multiple times over a period of several weeks. Nauplii can be collected directly from maturation tanks, but mated females are usually removed and placed in individual spawning tanks (from 100 to 500 liters), to facilitate maintaining water quality and nauplii collecting.
There is a widespread perception that postlarvae from females matured in the wild are superior and are generally preferred over postlarvae from ablated females, which are usually regarded as inferior in quality, growth and survival. There is evidence to refute this perception, and the reported lower quality of postlarvae from ablated animals may well be due to various other causes, including less than optimal treatment of broodstock at a time (after ablation) when there are increased energy requirements (proper nutrition) and disease susceptibility. Nauplii production from captive broodstock maturation varies considerably. As a point of reference, using the Oceanic Institute's High Health Broodstock Maturation System production can be about 1 million nauplii per broodstock (P. vannamei or P. stylirostris) over six months (Dr. Jim Wyban, High Health Aquaculture, Inc.; expert consultant on captive maturation of certified pathogen-free penaeid shrimp; personal communication).
Perspectives
Worldwide the production of penaeid seedstock - and the shrimp farming industry - is still totally dependent on wild broodstock (personal communication, Dr. Jim Wyban). This is a common thread among shrimp hatcheries, from the huge facilities in major shrimp farming countries like Indonesia and Thailand, to permanent and transient, backyard, family operated hatcheries in practically every shrimp farming country. This dependence is inherently unstable and potentially dangerous, as the number of wild shrimp stocks around the world that test positive for a variety of dangerous viruses increases. For example, wild P. vannamei stocks off Ecuador carry the Taura Syndrome Virus (TSV), and several P. monodon stocks in Asia are known to carry White Spot Virus (WSV).
The dependence on wild broodstock is more serious in Asia than in the western Hemisphere, as in Asia there has been less interest in captive reproduction of cultured broodstock because the existing system has so far fulfilled the needs of the growout industry (personal communication, Dr. Jim Wyban). In Latin America there has been more interest for cultured broodstock reproduction, partly because of the introduction of Pacific shrimp species into farms on the Atlantic side. A relatively recent survey of hatcheries in Central and South America (Wilkenfeld, 1992) indicated that 80% of the hatcheries depended completely on wild broodstock or spawners, while the remaining 20% rely to some degree on broodstock grown in their ponds. According to Bob Rosenberry (editor and publisher of the "Annual Report on World Shrimp Farming" and the "Directory of the Shrimp Industry in the Western Hemisphere"; personal communication), there are approximately 40 penaeid shrimp maturation facilities currently operating in the western Hemisphere, mostly in a handful of Latin American countries (about 12 in Ecuador). These facilities rely on captive broodstock and operate year round, producing mostly western white shrimp (P. vannamei) and also some blue shrimp (P. stylirostris). Some maturation facilities in Venezuela have reportedly achieved some degree of resistance against Taura Syndrome through broodstock selection over several generations in captivity, and are currently selling and exporting surplus nauplii and postlarvae production. At least one commercial supplier of nauplii in Ecuador is currently supplying "IHHNV-free" nauplii of P. vannamei and P. stylirostris from wild spawners screened using a commercially available IHHNV gene probe (personal communication, Dr. Jim Wyban). This probe is also in use at other hatcheries, and probes for other harmful viruses are under development. Gene probe technology holds much promise to screen out animals that carry unwanted pathogens, and is becoming a very important ally on the road to successful domestication and selection of disease resistant stocks of commercially important shrimp.
The recent development of high health captive populations of commercially important species has been a significant development to support the shrimp farming industry. The basic principles and technology for penaeid broodstock maturation are now widely applied and continually evolve and improve. But there are several areas in need of research, particularly on the understanding of endocrine pathways involved in the control of molting and reproduction (molt-inhibiting, gonad-inhibiting, gonad-stimulating and molting hormones) and how these respond to various environmental cues; and also genetic manipulation, selection and stock improvement. Artificial insemination is commonly done with P. vannamei and P. stylirostris when natural mating is inconsistent, and has been used in hybridization research with various species. Interspecific penaeid hybridization is another research area that is in its infancy, has enormous potential and will be discussed in a subsequent contribution.
(University of Miami Marine School, Miami, Florida, USA)
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