Principles of Genetics and Breeding

8.1.3.2 Diploidization

The haploid embryo generated by gynogenesis dies unless some special treatment is conducted, so apparently it is necessary for the embryo to become diploid by doubling its chromosomes. Polyploidization treatment can be performed at two stages.

• First method of polyploidization is by inhibiting meiotic phase II after insemination with sperm that has received a genetic inactivation treatment. When no crossing over occurs during meiotic phase I, maternal (it should be expressed grand maternal) or paternal chromatids of each chromosome remain in the ovum and the ovum develops into individuals containing pairs of the identical chromosomes (homozygotes=complete homogeneity in its gene makeup). On the other hand, the ovum containing exchanged homologous chromosomes which have arisen by crossing over does not develop into a homozygotic animal.
• Meiogynogenesis is achieved by inhibiting the extrusion of the second polar body. The resulting offspring are homozygous at a locus only if no recombination occurred. By determining the percentage of heterozygous offspring, the recombination frequency can be calculated. For some species, like rainbow trout, the residual heterozygosity is so high (up to 100% at some loci) that meiogynogenesis cannot be considered as an efficient inbreeding tool.

The second method is a polyploidization caused by the inhibition of cell division during the first cleavage. The original haploid set of chromosomes during meiotic phase I and II will be duplicated before the first cleavage, so that pairs of chromosomes after the first cleavage inhibition are homologous to each other irrespective of crossing over. It, therefore becomes a complete homozygote which generates genetically homologous eggs because genetic separation by meiosis does not occur. These eggs are genetically the same if they are polyploidized by gynogenesis, i.e. they are genetic copies of their mother, i.e. clones.

• Mitotic gynogenesis or endomitosis results in fully homozygous offspring, since it is achieved by inhibiting the first mitotic cleavage after duplication of the haploid genome.
• Homozygous inbred strains of genetically identical fish (clonal lines) can be obtained after two generations using this reproduction method. It has been achieved in zebrafish (Danio rerio),medaka (Oryzias latipes), common carp(Cyprinus carpio) Nile tilapia (Oreochromis niloticus) and Indian catfish (Heteropneustes fossilis).

Let us now consider the sex of offspring generated by gynogenesis. The most common sex methods are “XY” and “ZW” mechanisms. Two pairs of chromosomes are sex chromosomes that determine sex.

• In the case of the XY mechanism, the cells of the females contain two identical sex chromosomes, called X chromosomes (XX) and in males, the set of two sex chromosomes is composed of a single X chromosome and a smaller Y chromosome (XY).
• As a result of meiosis, all eggs contain one X chromosome, and two kinds of sperm are produced. Half contain an X chromosome, and half contain a Y chromosome. Fertilization of an X-bearing egg by X-bearing sperm result in an XX female zygote, and a fertilization of an X-bearing egg by a Y-bearing sperm results in an XY male zygote. Therefore about equal numbers of each sex are born.

What is the sex of the offspring generated by gynogenesis?.

• Since all eggs contain a X chromosome, the offspring will be XX female regardless of the stage at which the chromosomes are polyploidized.

The other mechanism of sex determination is the reverse of the XY mechanism, called the “ZW” mechanism.

• In this case, each female contains Z and W chromosomes, while each male contains two Z chromosomes. Therefore, two kinds of eggs are produced: half contain an Z chromosome, and half contain a W chromosome,while all sperm contain a Z chromosome.
• Fertilization of a Z-bearing egg by Z-bearing sperm result inZZ male, fertilization of a W-bearing egg by Z-bearing sperm result in ZW female. Application of gynogenesis to homogametic females results in all female progeny, while in the heterogametic female (ZW) offspring, 50% will be ZZ male and 50% WW female.

Table 1. Summary of results obtained with selected fishes by the induction of meiotic gynogenesis

Sl. No.	Species	Treatment	Shock	Time post fert. (min)	Treatment duration (min)	Surv ival (%)	Reference
1	S. gairdneri	Co60	HS 26oC	25	20	80	Chourrout and Quillet (1982)
2	Cyprinus carpio	UV irradiation 9000erg/mm2	CS 0-0.5oC	10-12	60	25	Taniguchi et al. (1986)
3	Cirrhinus mrigala	UV irradiation	CS 12o C HS 39o C	4 4	10 1	4 12-20	John et al. (1988)
4	Betta splendens	PS 7000	2.5	6	74	Pandian (1993)
5	C. gariepinus	UV irradiation	CS 5 ° C PS 55.00 Mpa HS 41oC	3 3 1	40 1.5 2	_ 68 _	Volckaert et al. (1994)
6	Cyprinus carpio	UV irradiation	HS 40o C CS 2-4oC	30 30	1.5 60	3.4 0.4	Bialowas (1995)
7	Cirrhinus mrigala	UV irradiation 580 m w/cm2	HS 39oC	4	2	2 to 50	Younus mia et al..(2001)
8	Heteropneustes fossilis	UV irradiation	HS 41oC	6	1	49.3	Godwin Christopher (2001)

Table. 2. Summary of results obtained with selected fishes by the induction of mitotic gynogenesis

Sl. No.	Species	Treatment	Shock	Time post fertilization (min)	Duration of treatment (min)	Survival %	Reference
1	O. niloticus	UV 300-310 muW cm-2 2 min,at 4o C	HS 41oC	27.5-30	3.5	-	Hussain et al. (1993)
2	O. niloticus	UV 300-310muW cm-2 2min,at 4o C	PS 630 kgcm-2	40-50	2.0	-	Hussain et al. (1993)
3	O. niloticus	UV irradiation	HS 42.5oC	22.5,25, 27.5,30	3 to 5	4	Myers et al. (1995)
4	Ictalurus punctatus	UV irradiation 0.08 or 0.12 J/cm2	PS 8000 psi	90	3	0.2	Goudie et al. (1995)
5	C. gariepinus	UV irradiation	HS 40.5oC	--	2	6.34	Varadi et al. (1999)
6	C. garie pinus	UV irradiation	HS 40oC	36-38	1	12	Galbusera et al. (2000)

HS - Heat shock

CS - Cold shock

PS - Pressure shock

Almost all diploids created by gynogenesis by the inhibition of meiotic phase II or the first cleavage are homozygotes, hence if recessive genes are contained,they express their effect. Gynogenesis, therefore, sometimes limits the lifetime or development of the organism due to the fact that two copies of recessive genes result in lethality or weak vitality. This decreased hatching rate or longevity can, however, be a favorable phenomenon in the respect that, in the normal case, recessive genes that are inherited by the next generation without expressing their effects cannot be completely eliminated from the population.

Thus in gynogenesis, harmful recessive genes are naturally eliminated by expressing lethality or can be removed by selecting individuals expressing harmful traits,allowing us to collect harmful-recessive-gene-free offspring, and from that, an extremely high life expectancy can be predicted.

Heterogeneous sperm are available since sperm do not contribute genetically to gynogenesis, i.e. any species of sperm are utilizable if they can penetrate eggs to give the stimulation needed for starting development. When the number of sperm are not available, species with maternal genes can be maintained by gynogenesis with exogenous sperm. Gynogenetic females were artificially produced in Indian major carps, tilapia, grass carp, silvercarp and zebrafish.