Field Document 3
December 1981
| FI: DP/IND/75/031 Field Document 3 December 1981 |
INDIA
FISH GENETICS, SELECTION AND HYBRIDIZATION
A report prepared for the
Intensification of Freshwater Fish Culture and Training Project
by
J. Bakos
Fish Culture Genetics Consultant
This is one of a series of reports prepared during the course of the UNDP/FAO project identified on the title page. The conclusions and recommendations given in the report are those considered appropriate at the time of its preparation. They may be modified in the light of further knowledge gained at subsequent stages of the project.
The designation employed and the presentation of the material in this document do not imply the expression of any opinion whatsoever on the part of the United Nations or the Food and Agriculture Organization of the United Nations concerning the legal or constitutional status of any country, territory or sea area, or concerning the delimitation of frontiers.
FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS Rome, 1981
Hyperlinks to non-FAO Internet sites do not imply any official endorsement of or responsibility for the opinions, ideas, data or products presented at these locations, or guarantee the validity of the information provided. The sole purpose of links to non-FAO sites is to indicate further information available on related topics.
This electronic document has been scanned using optical character recognition (OCR) software. FAO declines all responsibility for any discrepancies that may exist between the present document and its original printed version.
1.2 Fish Species Cultivated in India
2. SUMMARY DESCRIPTION OF WORK UNDERTAKEN
2.1 Establishment of the Genetical Laboratory
2.3 Development of Gynogenesis Technique for Carps
2.4 Identification and Maintenance of Land Races of Carps
3. CURRENT RESEARCH PROJECTS FOR FISH GENETICS
4. RECOMMENDED RESEARCH PROGRAMME AND ITS POSSIBILITIES
4.1 Methods and Phases of Research Work
4.1.1 Comparative Testing of Fish Species and Different Lines
4.1.2 Progeny Testing for Determining the Individual Breeding Value
4.1.3 Intraspecific Hybridization
4.1.4 Artificial Gynogenesis and Hormonal Sex Reversion
4.1.5 Application of Gynogenesis for Improving Productivity of Fish Species
4.1.6 Intergeneric Hybridization
4.2 List of Research Subjects Recommended for the Next Ten Years
5. EXPECTED RESULTS OF THE PROJECT RESEARCH
The Government of India, assisted by the United Nations Development Programme and the Food and Agriculture Organization of the United Nations, are engaged in the Intensification of Freshwater Fish Culture and Training Project (FI:DP/IND/75/031) at the Freshwater Aquaculture Research and Training Centre (FARTC) of the Central Inland Fisheries Research Institute (CIFRI) at Dhauli, Bhubaneswar, Orissa.
As part of the operation of the project, FAO assigned Dr J. Bakos as a Fish Culture Genetics Consultant for three months from 29 October 1980 with the following terms of reference:
To assist in establishing the fish genetical laboratory and in elaboration of studies on the selection of fish strains with better growth potential, meat quality, resistance to disease and longer breeding period;
To assist in the development of effective gynogenesis technique for carps;
To assist in identification and maintenance of land races of carps towards development of genetic pool.
During the assignment the consultant had detailed discussions with Dr V.R.P. Sinha, Project Director and Head of FARTC; Mr R.D. Chakrabarty, Head of the CIFRI Substation, Cuttack, and other scientists involved in breeding and genetical work. The Consultant wishes to thank all of them for their fruitful discussion. Thanks are also due to Dr Maharana of Cuttack Medical College for providing the cobalt 60 gamma ray instrument for the work.
The following fish species are commonly cultured in India and are thus the most important from the point of view of genetic selection and hybridization.
Indian major carps:
Catla (Catla catla H.)
Rohu (Labeo rohita H.)
Mrigal (Cirrhinus mrigala H.)
Exotic carps:
Silver carp (Hypophthalmichthys molitrix Val.)
Grass carp (Ctenopharyngodon idella Val.)
Common carp (Cyprinus carpio L.)
Other fish species which are important for fish culture are as follows:
Indian minor carps:
Reba (Cirrhinus reba H.)
Bata (Labeo bata H.)
Calbasu (Labeo calbasu H.)
White carp (Cirrhinus cirohosa Bloch)
Cauvery carp (Labeo kontius Fedon)
The Indian major carps are the endemic species in India and their contribution to the total fish production is considerable. Thus, it is essential to improve their genetical capabilities and also to maintain their gene bank. Equally important are the Chinese carps, which also need proper genetical selection.
The Genetical Laboratory consists of two rooms, one of them air-conditioned for under-taking biochemical investigations, such as electrofocussing, etc. The other room is for chromosomal investigations, gynogenesis and other genetic experiments. Another room will serve as a laboratory for selection and hybridization work. A fourth room will have provision for aquaria for rearing and keeping the progeny after gynogenesis and sex reversion. Here necessary equipment will be kept for marking, measuring and dissecting the fish. This room needs some drainage facilities for about 15 fibreglass tanks of different sizes.
After carefully examining the requirements of the genetical laboratory and the equipment already available, listed below is the additional equipment needed for the laboratory:
| (1) | Gel electrophoresis apparatus (both disc and slab) | 1 |
| (2) | Densitometer | 1 |
| (3) | Spectrophotometer | 1 |
| (4) | Microscope, Dialux 20 | 2 |
| (5) | Centrifuge, 500–5 000 rev/min | 1 |
| (6) | Centrifuge, refrigerated 10 000–13 000 g | 1 |
| (7) | Camera | 1 |
| (8) | Dissolved oxygen meter, electronic | 1 |
| (9) | Microscope, Olympus | 1 |
| (10) | Microscope, Stereo WILOM3 | 1 |
| (11) | Refrigerator 265 1 | 1 |
| (12) | Ultra-violet germicidal tube, 30 W Philips with extra tube | 1+1 |
| (13) | Refrigerator-deepfreezer 100 1 | 1 |
| (14) | Refrigerator-deepfreezer 500–1 000 (-20°C) | 1 |
| (15) | Meat mincer | 2 |
| (16) | Electronic mixer | 2 |
| (17) | Fibreglass tanks 1 000 1, 500 1, 200 1 capacity | 10 each |
| (18) | Electric aerator | 5 |
| (19) | Balance 0.5 g – 1 000 g | 1 |
| (20) | Dia slide projector | 1 |
| (21) | pH meter, electrical Philips | 1 |
Five different land races of catla, rohu, mrigal, silver carp, grass carp and common carp have to be maintained separately using a polycultural stocking system. As a safety measure, some specimens from all fish species must be maintained in two ponds in mixed conditions and permanently marked. This work requires 32 ponds (60 m2).
The fish fry produced with artificial propagation should be reared separately. The expected number of different populations yearly (three species with five various land races need 15 ponds of 150 m2), one intraspecific hybridization with polyallel crossings of five different females and males would need 25 ponds of 150 m2 and, for intergeneric hybridization, 5 ponds of 150 m2. Thus, altogether 45 ponds, 150 m2 each would be required for fry/fingerling rearing.
The area of testing ponds should be 2 000–5 000 m2 each. About 30 such ponds will be required for undertaking experiments with land races, hybrid populations, etc.
Further, about 10 ponds, 60 m2 each may be required for other genetical research. Therefore the total requirements of the ponds would be:
Because the major carps and Chinese carps breed only during the monsoon season, common carp was selected to be the test fish (common carp also breed during December-January) and carefully selected males and females were induced to breed with pituitary hormone injections. The sperm was irradiated with cobalt 60 and also some by ultra-violet ray. Stickness of the eggs was removed by urea and salt solution. During the embryonic development of eggs, a proper check was made of the development of the gynogenetic progeny. Identification was made of haploid and diploid embryos.
In certain countries a cobalt 60 gamma ray source in 100 000 Rad dose is given for irradiating the sperm. However, because of the difficulties in cooling the sperm here and also the duration of the cobalt 60 gamma ray, experiments were conducted to irradiate the sperm with the help of an available ultra-violet lamp. Appropriate duration of irradiation, intensity of the exposure and dilution of sperm were not known, so experiments were carried out to determine these. It was found out that the irradiation time should be 60 min, distance between the lamp and the sperm to be 12 cm, dilution 1:2 (dilution with frog Ringer) and the thickness of layer 2 mm. Mechanical shaking was used during the time of irradiation with a frequency of 120/min. Further experiments are necessary to find out the suitable method for cooling of sperm to 3–4°C during the irradiation.
The rearing of gynogenetic common carp progenies was carried out in plastic pools. They were initially given zooplankton collected from fish ponds for 28 days. After that they were given balanced artificial food consisting of sheep liver and heart and broiler chicken feed supplemented with vitamin A, B, C, D and E. The fry showed better growth rate than the normal fry of common carp. In order to have sex reversal in these fry, they have been fed with feed containing methyltestosteron. The result of the investigation will be known in due time. The above technique of gynogenesis has been carried out in collaboration with the counterpart scientists assigned to the consultant. Thus, these scientists are fully conversant with and trained to utilize this technique in other carps during their breeding season.
Collection of various land races of cultivated carp species (preferably at the fingerling stage) from different water systems of India is in progress, the first consignments of fish populations was to arrive in January 1981. Those carp species are also to be collected from Cuttack. Together with the Cuttack strain, five different land races of each species are suggested to be collected. FAO, in collaboration with the United Nations Environment Programme (UNEP), expects to develop a programme to preserve the genetic resources of aquatic organisms. Therefore, it would be better to have a link of the collection and maintenance of land races of carp along with the above programme.
After collection and arrival of fingerlings they need to be stocked in separate ponds. The various land races, as basic populations for research, have to be marked with fin clipping and operculum tagging. They should be reared in optimum conditions so as to achieve their best growth rate and early maturity. It is necessary to investigate the morphological/biochemical characteristics of different populations.
Selective breeding and hybridization
Studies on cytogenetical features of carp hybrids improving the strains of Indian major carps with crossing 1977–82.
Breeding of selected stock of grass and silver carps. Improved the stocks of Chinese carps 1977–81.
Studies on the morphology, growth and maturity of the hybrid between grass carp female and silver carp male 1978.
Hybridization between Labeo rohita × Cyprinus carpio; C. mrigala × C. Carpio; C. catla × C. carpio and C. catla × H. molitrix 1977–80.
In the field of genetics CIPRI attempted some preliminary experiments in interspecific and intergeneric hybridization, mainly for the purpose of improving the market quality of the fish (smaller head and deeper body, as in crosses between catla and rohu) and obtaining faster growth rates. Even though the results achieved are encouraging, it has not yet been possible to carry out adequate progeny testing due to a lack of facilities at the Cuttack farm. Genetic selection experiments to prevent inbreeding depression or to obtain strains with greater resistance to temperature variations common in tropical ponds, resistance to diseases and enhanced growth rates have not been attempted.
The aim of comparative testing is to determine and compare the most important characteristics which are closely correlated with the productivity of species. The different phases of comparative testing are as follows:
The groups for testing have to be produced with artificial propagation. One female and one male can be used which are chosen by phenotypical selection with the best growth rate and appearance.
The groups of progenies will be reared in separate ponds and after six months they need to be stocked together in a common pond with group marking (150–200 fish from each population at least). The stocking rate of testing ponds must be the same as in commercial fish farming/intensive polyculture. When the number of stocked fish from one population are less than 100 fish, the testing work has to be repeated three to four times with the same groups of fish.
It is necessary to use organic and inorganic fertilizers to fertilize the water and also to feed the fish.
The fish population reaching a marketable size need to be harvested. Proper measurement of every individual is required for evaluating different characteristics.
The qualitative characteristics, such as the colour, scales and the formation of fins are not closely correlated with the productivity, therefore the evaluation of those only for the purpose of morphological characterization of the populations has any importance.
From the quantitative characteristics one has to evaluate the viability, growth rate, food conversion, quantity of meat, quality of meat (protein and fat content) and the reaction to fish diseases. Later, by producing inbred lines, the fecundity and the oxygen demand tolerance also has to be examined.
The results obtained need to be verified with variance analysis.
The productivity of various groups can be compared through a multifactorial, “100 score” system by the four most important characteristics (like viability, growth rate, food conversion, quality of meat).
If the test is arranged in more ponds with replicates, standard population control has to be used in all ponds, which gives the possibility to compare the differences of environment among the ponds used. The common carp is suggested as a standard control considering its omnivorous feeding habits. The standard control can be produced in every year with a panmictic propagation, maintaining their quality and characteristics on the same level during a long time.
The results of pond testing experiments can be compared with the short laboratory tests and with the results of oxygen demand toleration (ODT) tests. The average of the above three methods gives a reliable final result of comparative testings.
The outstanding populations showing best characteristics will be used for the further selection and hybridization work.
The progeny testing is a suitable method to determine the individual breeding value of males and females after the productivity of their progenies.
The phases of the work are the following:
For the evaluation of males, the eggs of two females have to be fertilized with the sperm of chosen males separately, producing maternal half sister groups. Five or six males can be examined at the same time in their ten or twelve progeny groups from the two different females. For the evaluation of females one must follow the same system, using two different males.
The productivity of half sister groups are examined and compared by using the above mentioned comparative testing method (i.e., 4.1.1).
The average productivity of two groups originating from different females but from the same male shows the individual breeding value of the given male fish.
The best males and females in breeding value can be used for the intraspecific hybridization, or for improving the species in their characteristics.
The aim of intraspecific hybridization is to produce more productive hybrids by crossing different genotypes of females and males having the best results during the previous progeny testing. The phases of intraspecific hybridization are as follows:
Selection of various genotypes of the same species;
Inbreeding of the selected lines;
Polyallel crossing of the different lines;
Comparative testing of the F1 hybrid generation;
Propagation of the best hybrid producing parental lines for the fish seed production farms;
Producing three-line hybrids;
Producing four-line hybrids.
After the inbreeding of parental lines, the heterosis effect can be increased. The fastest method of inbreeding is the artificial gynogenesis.
As a result of intraspecific hybridization, the increasing productivity of fish species of about 15–25 percent can be expected.
The main purpose of gynogenesis is to produce inbred individuals having only maternal chromosomes for the further genetic work. For causing gynogenesis artificially, inactivated sperm can be used for fertilization of eggs, with a cold shock treatment after fertilization. For the inactivation of sperm a cobalt 60 gamma ray source or ultra-violet germicide lamp has to be used. The individuals of gynogenetic populations are females with diploid chromosome sets similar to the mother. The technique of artificial gynogenesis was demonstrated on the common carp. The method can be adopted for the other fish species with necessary modifications.
For producing gynogenetic inbred male populations, methyl-testosteron hormone feeding has to be provided for the females at a young age to influence the sex formation in the direction of the male. The technique of sex reversion was demonstrated with gynogenetic common carp. The same method can be used for all fish species which are able to feed with artificial food.
The inbreeding coefficient value (F) of the first gynogenetic population is already 0.90, which value can be reached only after 12 generations of sister-brother pairing.
For the purpose of increasing the inbreeding level, the gynogenesis can be applied from generation to generation. It is important to make a proper selection within the gynogenetic population to avoid the undesirable effect of inbreeding depression. The keeping and maintenance of gynogenetic inbred females and males requires more care than the normal ones because of their high breeding value and more sensitiveness to the environmental effects. Therefore, the location, rearing and keeping of this fish in small ponds is necessary, with a good water supply and other necessary conditions and to protect them from poaching. Some outstanding individuals can be kept in an indoor raceway system.
The gynogenetic inbred populations or individuals can be used for improving the species with their useful characteristics, to make inbred lines for intraspecific hybridization, for producing monosex fish populations and also for genetic examinations for particular purposes.
4.1.5.1 Producing and maintaining inbred lines for intraspecific hybridization
The greatest advantage of producing inbred lines is to obtain higher heterosis effect after the crossing of gynogenetic female and male lines.
The main phases of the work are as follows:
To select the best females for performance and morphological features.
Producing the first gynogenetic female generation (G1)
Rearing the gynogenetic fry suitably for producing sexually mature fish as soon as possible.
Selection by phenotype for the best growing and correct body formation.
Producing the second gynogenetic female generation (G2).
After dividing the G2 population into two parts, one part will be reared as a female
group and the other part has to be oversexed for producing gynogenetic males (G2 
).
Rearing the G2♀ and G2 groups suitably.
Brother-sister pairing can be used for producing the third gynogenetic generation
(G2♀ × G2 — G3♀). The purpose of brother-sister pairing is to avoid the chance
injuring of chromosomes.
Sex reversion in the G3♀ progeny group.
Brother-sister pairing for producing fourth gynogenetic generation (G2♀ × G3 — G4)
This scheme can be continued by the previous pattern. During all the phases the best female or male are selected according to their better constitution.
The inbred population produced can be used after phase five for intraspecific hybridization. After top crossing, good heterosis effect can be expected.
4.1.5.2 Producing semi-gynogenetic lines
The purpose of this work is to produce gynogenetic inbred male lines in cases when the hormone feeding is different, such as the case of silver carp and catla. The phases of work are as follows:
From population “A” selection to be made of the best females.
Producing the first gynogenetic generation (AG1♀).
Crossing the AG1♀ female with a normal A♂ male. The result obtained will be a semi-gynogenetic bisexual population (50% ♀ and 50% ♂).
Selection for the best females and males.
Producing the second gynogenetic generation (AG2♀)
Crossing of the AG2♀ female with the normal A♂ male. The scheme can be continued by the previous pattern. After the third phase the gene pool of population becomes homozygotic to about 60 percent.
4.1.5.3 Producing monosex fish populations with gynogenesis
The purpose of this work is: (i) to produce monosex female populations for pond fish culture because of their faster growth than that of the bisexual group of the same age; (ii) the stocking of monosex populations in natural waters like reservoirs or undrainable lakes; (iii) to develop inbred gynogenetic male lines is necessary for maintenance only in commercial fish producing farms or for fish seed production farms and pairing them with normal females; (iv) after solving the problem of deep freezing the sperm, one can collect and store the sperm of inbred gynogenetic suitable males, so as to use it for producing monosex populations in other farms.
The phases of the work are as follows:
To select female from a male line
Producing the first gynogenetic population (G1 ♀)
Producing a gynogenetic male population with hormonal sex reversion (G1 )
Using the selected males for top-crossing by pairing them with normal females.
The progenies will be all females which may show improved characteristics.
Intergeneric hybridization consists of crossing fish belonging to different species. The aim of producing intergeneric hybrids is as follows:
To unite the useful characteristics of two different species in one of the new forms.
Producing a new construction in consequence of the heterosis effect.
Using intergeneric hybridization, the following results can be expected:
New construction with new feeding habits for composite fish culture or for water reservoirs
Better growth rates and survival rates
More restful behaviour, having better tolerance for reduced levels of oxygen (silver carp × bighead)
Better quality of meat (common carp × silver carp; rohu × catla)
Monosex triploid grass feeder fish (grass carp × bighead)
Better tolerance of water salinity for brackishwater fish culture.
In the field of intergeneric hybridization the scientists of India have achieved good results (see Section 3).
The phases of intergeneric hybridization are the following:
Studying the fish species characteristics; behaviour and genetic structures
Analysis of chromosome sets and karyotypes
Crossing the species, using artificial propagation
Rearing the hybrids in the most suitable environment for their expected habitat
Comparative testing of hybrids and the parental forms
Selection of the most suitable individuals
Artificial propagation experiments on the F1 hybrids
Stabilizing the new characteristics with inbreeding or back-crossing with the closer parental species which are similar in their main characteristics to the new hybrids
Describing the new hybrids' morphological, biological and economic characteristics.
4.1.7.1 Biochemical polymorphysm investigations
These investigations are aimed at finding and using genetic markers for characterization and identification of fish populations and individuals. Such markers are the enzymes and proteins, including transferrins. The method is very important for characterization of species, land races and hybrids. Also this is useful in testing the success of gynogenesis in fully scaly fish species. The instrument for this method is the polyacrilamid gel-electrophoretic equipment which was ordered.
4.1.7.2 Chromosome investigations
These investigations have the aim of characterization of fish species and hybrids to study the possibilities of intergeneric hybridization. The method of chromosome investigation is well known by the scientists but some technical details have to be improved. These investigations will be also important in the future research programme with the aim of polyploidization.
1. To develop practical methods of artificial gynogenesis and hormonal sex reversion of catla, rohu and mrigal to produce highly inbred parental lines. Intraspecific hybridization of catla, rohu and mrigal, with crossing gynogenetically inbred female and male lines, to compare the productivity of the different populations.
2. To develop practical methods of artificial gynogenesis and hormonal sex reversal of silver carp and grass carp to produce highly inbred parental lines. Intraspecific hybridization of silver carp and grass carp with crossing gynogenetically inbred female and male lines. To compare the productivity of the different populations.
3. To develop practical methods of artificial gynogenesis and hormonal sex reversion of common carp to produce highly inbred parental lines. Intraspecific hybridization of common carp with crossing gynogenetically inbred female and male lines.
4. Fish chromosome investigations on Indian major carps and cultivated exotic carp species.
5. Biochemical polymorphism investigations on Indian major carps and the cultured exotic carp species to determine the transferrin and isoenzyme polymorphysm of different genotypes with a view to selection and hybridization of cultivated species. Intergenetic hybridization among:
6. To develop practical methods of producing polyploid and androgenetic fish populations in Indian major carps and the common carps.
7. Establishment and maintenance of the living gene pool of Indian major carps, collecting the different land races, elaborating practical marking systems, propagating for renewal of populations every five years and using them in the genetic and selection work.
8. Spreading the new fish hybrids into the commercial fish farms and fish seed producing farms in the form of inbred female and male lines.
Some results of project research will provide a scientific basis for future research but other results should assist the producing sector to develop better results in actual fish farming. As a result of chromosome and biochemical investigations one can become acquainted with the genetic structure of cultivated fish species and obtain additional information to determine the further directions of the work of selection and hybridization.
By the comparative testings and progeny testings the best populations and individuals are selected in terms of their productivity. Using the best strains, the population can be improved, not only in the Institute but also in the fish farms. By improving productivity of species with intraspecific hybridization, on the basis of Hungarian experience, the following results can be expected: in the survival rate + 20%, growth rate + 20%, conversion of food + 15% and increasing percentage of eatable meat + 2–3%.
The monosex female populations provide 10–15% higher growth than the bisexual mixed ones in the same environment. By artificial gynogenesis and hormonal sex reversion inbred female and male lines can be produced, and from their heterosis hybrids 30–40% higher productivity can be expected. By improving the fish species by the above mentioned methods, the productivity of composite fish culture should be increased about 15–20% without any extra financial investment.
Rearing of the highly valuable fish fry produced with gynogenesis and sex reversion can be accomplished only in overflow, raceway systems. The presently used plastic pool system is unsuitable for keeping and feeding fish for long periods. Therefore, it is necessary to transform the fish aquarium laboratory into a modern overflow aquarium room urgently.
One of the basic works of genetics and selection is the artificial propagation of selected fish. FARTC has a plan to build a modern, multipurpose fish hatchery. The hatchery should be constructed as soon as possible to enable many important research projects on genetics and breeding to be undertaken.
It is recommended to import and acclimatize the Hungarian strain of common carp, which has been selected for its good growth rate, good food conversion and longer period of sexual maturation. It would be important to examine this common carp in composite fish culture conditions.
