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By Kevin Fitzsimmons
Department of Soil, Water and Environmental Science
University of Arizona, Tucson, Arizona, 85706

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Kevin Fitzsimmons

University of Arizona

One of the basic factors of tilapia aquaculture is that male fish grow bigger and faster than the females.  Also, in order to avoid unwanted spawning in a production unit, all-male populations are preferred.  There are several methods used to skew sex ratios and increase the percentage of males in a population.

The first method developed was to simply cull through a population, discard the females and keep the males.  This system is obviously wasteful and inefficient.  In the 1960’s and 70’s,  Israeli scientists discovered that certain hybrid crosses resulted in skewed sex ratios favoring males.  There are several theories regarding the genetic factors involving the number and location of sex genes on particular chromosomes.  The use of hybrid crosses is still one of the primary methods of producing mostly male populations.  The drawback to this method is that two separate broodlines must be maintained.  The crossing must be done very carefully and meticulous records should be kept to insure that the parent species are kept pure.  Also, usually only one sex from each species is used for any particular cross because the reciprocal cross (using the other sex from each species) is not as successful.  Another problem is that the number of young produced is rarely as high as a single species spawn.  Therefor, to maintain a commercial scale hatchery will usually require significant resources and staff.

The more common method of generating mostly male populations is through the use of steroids fed to sexually undifferentiated fry.  Newly hatched tilapia are still developing their gonads.  Even though they are determined genotypically their phenotype, or morphological characteristics can still be altered.  By exposing the fish to forms of testosterone or estrogen, the gonad can switched.  Typically the desire is to produce all males, so methyltestosterone is included in the diet for several weeks when the fish start eating.  Other hormones have been tested and sex-reversal can also be achieved by immersion in a solution.  The hormones cause the gonads to develop as testes instead of ovaries and the fish will also take on male morphological characteristics. The hormone is only needed during the first month and after that the fish are feed normally for the rest of their lives.  Using this technique farms can produce populations of greater than 90% male fish.  These populations grow faster than equivalent populations of mixed sex fish and have significantly less reproduction in the growout systems.

A novel variation on this scheme is to feed young fish estrogen.  This results in a population of all female fish.  The morphological female but genetic male fish are then reared to maturity and then mated to normal male fish.  The resulting fry have a male father and a male mother and thus will all be male.  These young have never been treated with any hormone.  Of course this technique requires several years to develop the stocks and extensive progeny testing to determine which fish produce the all male young.  Even more complicated breeding plans have been developed to fix breeding lines of male with two male chromosomes that will always produce male offspring.

Additional research is underway to study the basic physiology of sexual differentiation.  How the genes are turned on and off, how the genetic complement from each of the parent fish contribute to the genetic makeup of the young and how environmental and chemical stimuli affect the development of the gonads.  By better understanding these phenomenon we hope to develop more efficient methods of directing the sexual development of the fish.

University of Arizona

One of the great advantages of tilapia for aquaculture is that they feed on a low trophic level.  The members of the genus Oreochromis are all omnivores, feeding on algae, aquatic plants, small invertebrates, detrital material and the associated bacterial films.  The individual species may have preferences between these materials and are more or less efficient depending on species and life stages in grazing on these foods.  They are all somewhat opportunistic and will utilize any and all of these feeds when they are available.  This provides an advantage to farmers because the fish can be reared in extensive situations that depend upon the natural productivity of a water body or in intensive systems that can be operated with lower cost feeds.

In extensive aquaculture, the fish will be able to grow by eating algae and detrital matter and the farmer can grow more fish in a given area because the fish are depending directly on the primary productivity of the body of water, primary consumers.  Fish which feed on a higher trophic level, eating larger invertebrates or small fish, are secondary consumers and a system can only support a fraction of the biomass of secondary consumers compared to primary consumers.

In intensive systems, tilapia have the advantage that they can be fed a prepared feed that includes a high percentage of plant proteins.  Carnivorous fish require fish meal or other animal proteins in their diets, which in general are more expensive than plant proteins.  Nutritional studies which substitute plant proteins supplemented with specific amino acid supplements may lower costs, but still not to the level that can be achieved with tilapia diets.

Complete diets are used in systems that cannot provide any dependable nutrition.  This would include intensive recirculating systems, cages placed in water with low productivity and even heavily stocked ponds that do not provide enough nutrition for all the fish in the system.  Supplemental diets will provide only portions of the nutritional demands of the fish, with the assumption that they will get most of the nutrients from the growing system.  Supplemental diets are usually much less expensive than complete diets and usually high in carbohydrates.  Some simple supplemental diets serve a dual purpose of fertilizing the pond as well increasing productivity.  Considerable research has been conducted on complete diets and on fertilization programs for natural and man-made water bodies.  Development of supplemental diets directed to specifically provide limiting nutrients is a growing area of research.

Tilapia exhibit their best growth rates when they are fed a balanced diet that provides a proper mix of protein, carbohydrates, lipids, vitamins, mineral and fiber.  Jauncey and Ross (1982), El-Sayed and Teshima (1991) and Stickney (1996) provide excellent reviews that examine the details of tilapia nutrition.   The nutritional requirements are slightly different for each species and more importantly vary with life stage.     Fry and fingerling fish require a diet higher in protein, lipids, vitamins and minerals and lower in carbohydrates as they are developing muscle, internal organs and bone with rapid growth.  Sub-adult fish need more calories from fat and carbohydrates for basal metabolism and a smaller percentage of protein for growth (Table 1).  Of course the absolute amount the fish is eating will still be increasing as the fish is much larger.  Adult fish need even less protein, however the amino acids that make up that protein need to be available in certain ratios (Table 2). Feed formulators will adjust protein sources to fit the desired pattern of amino acids through the growth cycle.    Broodfish may require elevated protein and fat levels to increase reproductive efficiency (Santiago et al. 1985; Chang et al 1988).

 Table 1. Typical protein requirements for tilapia

1.    First feeding fry 45 - 50 %
2.    0.02 - 2.0 g 40%
3.    2.0 - 35 g 35%
4.    35  - harvest 30 - 32 %

Table 2. Essential Amino Acids in experimental tilapia diets at the University of Arizona.

Essential Amino Acids          g/kg diet        % of protein
Arginine                            15   7.5
Histidine          5   2.3
Isoleucine      9   4.3
Leucine    15   7.0
Lysine    16   5.0
Methionine      5   (74% of Cysteine)    1.7
Phenylalanine    15   4.5
Threonine    10   3.6
Tryptophan      2   1.0
Valine    12   5.8

In general, the lipid requirements for fish under two grams represent 10% of the diet. This decreases to 6-8% from two grams to harvest.  The lipids should contain both omega 3 and omega 6 fatty acids.  Each fatty acid should represent 1% of the diet, although some reports suggest that fish grow better with a higher proportion of omega 6 to omega 3.   The fiber component is usually the reciprocal of the lipid content.  That is starting at 6-8% in small fish up to 35g and increasing to 10% above 35g.  Carbohydrates usually represent less than 25% of the diet for fish under a gram and increases to 25 - 30% for fish greater than a gram up to harvest.

Carbohydrates are often supplied by the least expensive ingredients in the diet. Corn, wheat, rice and a number of agricultural byproducts are typical carbohydrate sources.  The ratio of energy supplied by lipids and carbohydrates to the proteins available in the diet is often a critical measure.   Shiau (1997) provides a comprehensive review of carbohydrate and fiber utilization in tilapia.

Vitamins and minerals are critical to proper nutrition in tilapia and considerable research has been conducted to determine these requirements (Watanabe et al. 1997; El-Sayed and Teshima 1991; Roem, et al. 1990; Jauncey and Ross 1982).  Commercial premixes are available which allow feed makers to purchase a whole group of micronutrients rather than attempting to determine how much is available from the productivity of the system and the other ingredients (Table 3).

Table 3.  Vitamin  and mineral mix used in University of Arizona tilapia diet.

     (recommended amounts before pelletizing)

Vitamins

 mg/kg                     I. Units

Thiamin               11

Folic acid          5

Riboflavin  20

Vitamin    B12       0.01

Pyridoxine  11

Choline 275

Panthothenic acid   35

Nicotinic 88

Ascorbic acid (C) 375

Vitamin K         4.4

Vitamin  A  4,400

Vitamin  D3  2,200

Vitamin  E        66

Minerals          g/kg

Calcium      Ca   3.0

Phosphorus   P    7.0

Magnesium    Mg  0.5

Iron         Fe   0.15

Zinc         Zn   0.20    (Note: Should not be above 0.3 (300 ppm)

Copper       Cu   0.003

Manganese    Mn   0.013

Selenium     Se   0.0004

Iodine       I    0.001

Feed Formulations

Feed manufacturers will adjust the mix of ingredients to create what are called “Least Cost Feed Formulations”.  These are formulations that use spreadsheet and database programs to examine the nutritional characteristics of many ingredients at the same time.  The program can then select the mix of ingredients that meet all the nutritional requirements at the lowest manufacturing cost.  These feeds will then meet the “Guaranteed Analysis” on the manufactures label, which tells any purchaser of the feed what they can expect from the feed.

This is meant to be only a short introduction to tilapia nutrition.  Specific nutritional needs vary by species, age of fish, production system, and salinity.  A wealth of information is available and feed manufacturers have developed considerable expertise.  Tilapia nutrition is critical to further increases in efficiency and profitability for the small producer growing for personal consumption and the large producer in international trade.

References

Chang, S.L., Huang, C.M. and I.C. Liao  1988.  Effects of various feeds on seed production by Taiwanese red tilapia.  In Proceedings of the 2nd International Symposium on Tilapia in Aquaculture.  Pullin, R.S.V., Rhukaswan, T., Tonguthai, K. and Maclean, J.L., Eds.  ICLARM, Bangkok.

El Sayed, A.F.M., and S.I. Teshima  1991.  Tilapia nutrition in aquaculture.  Reviews in aquatic sciences. 5 (3-4):247-265.

Jauncey, K. and  B. Ross  1982.  A Guide to Tilapia Feeds and Feeding.  University of Sterling, Scotland.

Roem, A.J.,  R.R. Stickney,  and C.C. Kohler  1990.  Vitamin requirements of blue tilapias in a recirculating water system.  Progressive Fish Culturist 52:15-18.

Santiago, C.B., Aldaba, M.B., Aubuan, E.F. and M.A. Laron   1985.  The effects of artificial diets on fry production and growth of Oreochromis niloticus breeders.  Aquaculture, 47:193.

Shiau, S.Y. 1997.  Utilization of carbohydrates in warmwater fish - with particular reference to tilapia, Oreochromis niloticus x O. aureus.  Aquaculture 151:79-96.

Stickney, R.R.  1996.  Tilapia update, 1995.  World Aquaculture 27(1):45-50.

Watanabe, T.,  Kiron V. and S. Satoh  1997.  Trace mineral in fish nutrition.  Aquaculture 151:185-207.

Basic tilapia genetics

We are at an early stage in the genetic domestication of tilapia. During the approximately 40 year history of intensive culture, the genetic resources of tilapia have been poorly managed. The genetic problems now manifesting themselves are of several kinds. First is the loss of pure species through mismanagement of interspecific hybridization (McAndrew 1993), a technique which has been used to produce all-male fry which have a higher growth rate in production systems (Hickling 1960; Hulata et al.1983). One popular commercial strain is thought to contain genes from as many as four species (McAndrew et al. 1988). A second problem is high levels of inbreeding depression. Primary collections of wild broodstock frequently consisted of a small number of individuals. These were serially distributed, so that genetic problems have been passed from country to country, and farm to farm. Eknath et al. (1993) compared four strains farmed in the Philippines with four strains newly isolated from wild populations in Africa. The best performing strains were those most recently isolated from nature, consistent with the idea that domesticated strains suffer from inbreeding depression (Tave and Smitherman 1980; Hulata et al. 1986; Teichert-Coddington and Smitherman 1988). A survey of 18 microsatellite DNA markers in several commercial strains found some strains with heterozygosities less than 10% of that found in wild strains (Kocher et al., unpubl.). In addition to inbreeding, it is likely that negative selection for growth rate has occurred during the propagation of many stocks. Finally, there is evidence for contamination of genetically improved strains by introgression from feral species (Macaranas et al. 1986).
In recent years attention has focused on a single species, O. niloticus, and research has begun to overcome some of the main problems associated with farming this species (Pullin and Capili 1988; Tave 1988). Large scale genetic improvement programs have been established for O. niloticus in Asia (Eknath et al. 1993) and genetic methodologies to control sex have now resulted in the reliable production of all-male fry to help overcome the problems associated with excessive fry production in on-growing ponds (Mair et al. 1995). Future research will aim to improve the performance and expand the environmental tolerance of this species into areas of lower temperatures and higher salinities. These developments will require a greater use of genetic markers for the management of broodstock, identification of loci controlling quantitative traits (QTLs) and the development of superior strains through marker-assisted selection. Development of tilapia breeds is at a point where tremendous gains could be made with a relatively small technical input.

Tilapia genome project

To support genetic improvement of tilapia, an international effort has begun to compile a map of the O. niloticus genome (Kocher, 1997). Until recently, only a few genes had been mapped in tilapia. Hussain et al. (1994) used meiotic gynogens (diploid progeny produced by suppression of the 2nd meiotic division) to map gene-centromere distances for six allozyme loci and two color loci. The karyotypes of the various tilapia species are highly similar, consisting of 22 pairs with no morphologically distinct sex chromosomes. In fact only two pairs are recognizable, the remaining 20 being similar in size and acrocentric morphology (Majumdar and McAndrew, 1986). At the molecular level, the genome size of several species has been measured at around 1pg (1,000Mb), about one-third the size of many mammalian genomes.
Recent work has focused on mapping DNA polymorphisms, including microsatellites (e.g. Lee and Kocher, 1996) and anonymous fragment length polymorphisms (AFLPs; Vos et al. 1996). These markers are highly polymorphic, and easily scored from small tissue biopsies (e.g. fin clips). We have recently completed a first genetic linkage map for a tilapia (Oreochromis niloticus) (Kocher et al., 1997). The segregation of 62 microsatellite and 112 AFLP polymorphisms was studied in 41 haploid embryos derived from a single female. We have identified linkages among 162 (93.1%) of these markers in a map which spans approximately 700 centimorgans. We anticipate that the final length of the genetic map for this species will be just over 1000 cM.
This linkage map is complete enough to begin mapping genes responsible for production traits. For example, if we wanted to combine genes from two strains to produce a superior line, we might hybridize the strains and use the DNA markers to track the segregation of chromosomal segments in the F2 progeny. Desirable genes could be tracked and selected using linked DNA markers from the map.

Transgenic tilapia

Another approach to genetic improvement is the insertion of new genes or gene constructs into the genome (transgenics). Many of the technical hurdles in the production of transgenic animals have been surmounted, and the introduction and expression of foreign genes is becoming routine. At least two groups have produced introduced additional copies of the growth hormone gene into tilapia and found an enhanced growth response (Martinez et al. 1996; Rahman et al. 1997). It is not yet clear whether these insertions are stable over many generations, or what additional effects they may have on the physiology of the animal. To be useful, these constructs will have to be carefully maintained in a selective breeding program. Application of this technology to other
production characteristics awaits the identification of genes responsible for these traits.

Acknowledgments

Research on the tilapia genome has been funded by the USDA NRICGP (#94-37205-1033).

References

Eknath, A.E., M.M Tayamen, M.S. Palada-de Vera, J.C. Danting, and R.A. Reyes, 1993. Genetic improvement of farmed tilapias: the growth performance of eight strains of Oreochromis niloticus tested in different farm environments. Aquaculture 111: 171-188.

Hickling, C.F. 1960. The Malacca Tilapia hybrids. J. Genetics 57:1-10.

Hulata, G., G. Wohlfarth and S. Rothbard. 1983. Progeny-testing selection of tilapia broodstocks producing all-male hybrid progenies--preliminary results. Aquaculture 33:263-268.

Hussain, M.G., B.J. McAndrew, D.J. Penman and P. Sodsuk., 1994. Estimating gene-centromere recombination frequencies in gynogenetic diploids of Oreochromis niloticus L., using allozymes, skin colour and a putative sex-determination locus (SDL-2). pp. 502-509 in A.R. Beaumont (ed). Genetics and Evolution of Aquatic Organisms. Chapman and Hall.

Kocher, T.D. 1997. Tilapia Status Report. Proceedings of the Aquaculture Species Genome Mapping Workshop, May 18-19, Dartmouth, Massachusetts.

Kocher, T.D., W.-J. Lee, H. Sobolewska, D. Penman and B. McAndrew 1997. A genetic linkage map of a tilapia (Oreochromis niloticus). Submitted.

Lee, W.-J., and Kocher, T. D. 1996. Microsatellite DNA markers for
genetic mapping in the tilapia, Oreochromis niloticus, J. Fish Biology 49:169-171.

Macaranas, J. M., N. Taniguchi, M. J. R. Pante, J. B. Capili and R. S. V. Pullin, 1986 Electrophoretic evidence for extensive hybrid gene introgression into commercial Oreochromis niloticus (L.) stocks in the Philippines. Aquaculture and Fisheries Management 17: 249-258.

Majumdar, K.C. and B.J. McAndrew. 1986. Relative DNA content of somatic nuclei and chromosomal studies in three genera, Tilapia, Sarotherodon, and Oreochromis of the tribe Tilapiini (Pisces, Cichlidae). Genetica 68:175-188.

Martinez, R., M.P. Estrada, J. Berlanga, E. Guillen, O. Hernandez, 1996. Growth enhancement in transgenic tilapia by ecotopic expression of tilapia growth hormone. Mol. Mar. Biol. and Biotech.5:62-70.

McAndrew, B. J. 1993. Sex Control in Tilapiines. pp. 87-98 in Recent Advances in Aquaculture IV , edited by R.J. Roberts and J. Muir. Blackwell Scientific Publishing.

McAndrew,B.J., F.R. Roubal, R.J. Roberts, A.M. Bullock and I.M. McEwen. 1988. The genetics and histology of red, blond and associated colour variants in Oreochromis niloticus. Genetica 76:127-137.

Pullin, R.S.V. and Capili, J.B. 1988. Genetic improvement of tilapias: problems and prospects, pp. 259-265 in The Second International Symposium on Tilapia in Aquaculture; edited by R.S.V. Pullin, T. Bhukaswan, K. Tanguthai, and J.L. Maclean. ICLARM Conference Proceedings 15, Manila.

Rahman, M.A., A. Smith, R. Mak, H. Ayad and N. Maclean. 1997. Expression of an exogenous piscine growth hormone gene results in enhanced growth in transgenic tilapia (Oreochromis niloticus). Sixth International Symposium on Genetics in Aquaculture, Stirling, U.K.

Tave, D., 1988 Genetics and breeding of tilapia; A review, pp. 285-293. In: The Second International Symposium on Tilapia in Aquaculture; edited by R.S.V. Pullin, T. Bhukaswan, K. Tanguthai, and J.L. Maclean. ICLARM Conference Proceedings 15, Manila.

Tave, D. and R.O. Smitherman, 1980 Predicted response to selection for early growth in Tilapia nilotica. Trans. Amer. Fish. Soc. 109: 439-445.

Teichert-Coddington, D.R. and R.O. Smitherman, 1988 Lack of response by Tilapia nilotica to mass selection for rapid early growth. Trans. Amer. Fish. Soc. 117: 297-300.

Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M., and Zabeau, M., 1995. AFLP: a new technique for DNA fingerprinting, Nucleic Acids Res, 23, 4407-4414.