Principles of Genetics and Breeding

8.1.4 Polyploidy

A promising biotechnological tool for increased production of food from aquaculture and creation of sterile organisms is polyploidy. Polyploidy refers to a genetic state that can be produced artificially in fish and shellfish through manipulation of embryos. Polyploid individuals have extra sets of chromosomes beyond the normal 2, with triploids having 3 and tetraploids having 4. Although it is a lethal state for mammals and birds,polyploidy has shown some promising results in the field of aquaculture.

• Triploid fish and shellfish are viable and tend to be sterile due to a lack of gonadal development. This sterility allows for reproductive energy to be diverted toward somatic growth, resulting in higher growth rates for some triploid individuals.
• Although triploidy has been highly effective for enhancing growth in shellfish, results thus far in fish have shown variable, conflicting results, with reports of triploid fish growing slower, at the same rate, or faster than diploids.
• When meiotic phase II is inhibited after insemination of a normal egg with a normal sperm, the female nucleus with 2 sets of chromosomes and male nucleus with one set of chromosomes combine, yielding a triploid with 3 sets of chromosomes, while if the first cleavage is inhibited, a tetraploid with 4 sets of chromosomes by chromosome replication can be obtained.
• In polyploidy plant that contains more sets of chromosomes the plant itself or the fruits become larger, and the same phenomenon can be expected in animals. But in fish, the tendency to become large according to polyploidization and each volume is in proportion to ploidy. Therefore these traits can be used to identify the ploidy.
• The triploid condition often causes sterility. Reproductive organs of normal aquatic organisms are large often accounting for more than 30% of the gross weight. Therefore, if sterilization by triploidization can be achieved, the energy used for formation of these reproductive organs can be applied to body growth.
• Sterile triploids of exotic species could be safely used where the introduction of such exotic species could otherwise lead to competition with native fish and suppression of the later.
• The induction of triploidy is a very useful technique to regulate and control populations of uncontrolled breeding species like tilapias, and the grass carp, which had been introduced into the exotic habitats. One such example is the introduction of grass carps into the reservoirs of the USA to reduce unwanted growth of macrophytes.

Table.3. Induced triploidy reported in various fishspecies.

Sl. No.	Species	Meiosis interruption	Time post-fertn. (min.)	Duration (min.)	Ploidy %	References
1	Cyprinus carpio	Cold shock 0-2 ° C	1-9	45	60	Gervai et al. (1980)
2	Rhodeus ocellatus	Cold shock 0-0.2 ° C	5	60	95	Ueno and Arimoto (1982)
3	Ictalurus punctatus	Cold shock 5 ° C	5	60	100	Chrisman et al. (1983)
4	Clarias gariepinus	Cold shock 5 ° C	2-4	40	80-100	Richter et al. (1987d)
5	C. gariepinus	Cold shock 5 ° C	3	40	95	Henken et al. (1987b)
6	Oreochromis mossambicus	Heat shock 42 ° C	2.5	3	100	Varadaraj and Pandian (1990)
7	Labeo rohita	Heat shock 42 ± 0.5 ° C	7	1-2	12	Reddy et al. (1990)
8	Clarias macrocephalus	Cold shock 5 ° C	2	30,45,60	100	Vejaratpimol and Pewnim (1990)
9 10	Cyprinus carpio O. niloticus	Heat shock 40 or 41 ° C Pressure shock 8000 psi	6 9	2 or 1.5 2	80-100 -	Recoubratsky et al. ( 1992) Hussain et al. (1996)
11	O. aureus	Heat shock 41 ° C	3	4	100	Chang and Liao (1996)
12	Heteropneustes fossilis	Heat shock 40ºC	4	2	76	Godwin Christopher (2001)
13	C. carpio	Heat shock 40 ° C	1-3	1.5	-	Basavaraju et al. (2002)
15	Cirrhinus mrigala	Heat shock 40ºC	5	1	80	Karl Marx et al. (2003)

• Induction of triploidy basically involves applying shock treatment by heat, cold, pressure or chemicals to eggs soon after the sperm entry when the eggs are about to undergo the mitotic phase of meiosis.
• The shock treatment suppresses the separation of sister chromatids (anaphase II) thereby preventing the second polar body formation inducing the diploid condition instead of maintaining the haploid. With subsequent fusion of the diploid egg and haploid spermatozoa a triploid zygote results.
• The gonad of a triploid male is generally less developed than that of a diploid male. Spermiogenesis of triploids is disturbed by the irregular pairing of the three sets of chromosomes and if spermatogonia develop, they are aneuploid.
• Triploid females have under developed ovaries, resulting in a much higher carcass index than diploid females. Maturation is completely suppressed. Triploid males have partially developed testes and also a good carcass index.
• In the course of sexual maturation, triploids (especially females) of numerous species have shown to excel diploids in growth and some taste characteristics. Triploid individuals are expected to be functionally and endocrinologically sterile due to their meiotic inhibition of gametogenesis and lack essential steroid hormones to support gonadal growth.

Triploidy has been induced in different species of fish through thermal shocks ; by use of chemicals such as colchicine, nitrous oxide, high pH, by high calcium and by pressure shocks.

• Svardson (1945) produced triploid salmonid fish embryos through coldshock.
• Swarup (1959) was the most successful of earlier workers, producing triploid fish which were reared to full size. Diploid and triploid sticklebacks (Gasterosteus aculeatus) differed in certain body features but were similar in growth rate and viability.
• Triploid grass carp (C. idella) has been produced by means of cold shock at 5-7°C for 25-30 min, 2-4.5 min after fertilization for use in controlling aquatic vegetation, without the risk of causing environmental disturbances.
• The assessment of triploid C. carpio for culture was done by Cherfas et al. (1994) who found that most triploid males and females had undeveloped gonads and were sterile. Triploids grew slower than their diploid sibs.
• Flajshans (1996) induced triploidy in C.carpio and Tinca tinca and the study revealed insignificant differences in live weight and dressing performance in triploid common carp against its diploid siblings while triploid tench recorded 13.52–23.66 % higher live weight against its diploid siblings.
• The effect of heat shock in C. carpio var. communis was assessed by Reddy et al. (1998). Differences in growth rate between diploids and triploids were noticed from fifth to sixth months of age onwards. The growth rate of triploids was observed to be significantly higher than the diploids sibs (P<0.01) till nine months of age.
• Sarangi and Mandal (1994) established an efficient protocol for generating tetraploid, triploid and gynogenetic stock in three Indian major carps under the island conditions of Andamans. Karal Marx et al. (2003) produced triploid mrigal, Cirrhinus mrigala by applying heat shock at 40^oC for 1 min, 5 min after fertilization.
• The triploid transgenic females could offer a good option for aquaculture purposes because they showed rapid growth performance over the normal wild-type tilapias with the advantage of sterility to ensure non hybridization and non contamination with the local gene pool.

The advantage of triploid fish compared to normal diploid fish are that

1. they are generally sterile and therefore useful for stocking natural bodies of water where population control is desirable and
2. they may grow faster at and after sexual maturity (in triploids 20-30% of the energy utilized for normal gonadal development is diverted to somatic growth).

Triploid salmon are widely cultured in Canada and on a smaller scale also in Japan.These are produced either by direct triploidization induction or by breeding tetraploid (XXXX) females with diploid (XX) males.