Principles of Genetics and Breeding

Induction of gynogenesis in fish

Gynogenesis is the process of embryonic development with solely maternal genome and without paternal genetic input. The main objective of gynogenesis is to produce highly homozygous inbred lines in much shorter time than through the conventional breeding process. These gynogen lines can be top-crossed with the heterozygous stock to achieve heterosis effect. Gynogenesis can be artificially induced by eliminating/denaturing the genetic material (DNA) of the milt through irradiation either by UV or gamma rays and activating the eggs with irradiated milt. Diploidy is restored by giving thermal or hydrostatic pressure shock. Retention of diploidy in a haploid gynogen egg can be done in two ways.

1. By suppressing metaphase II in the second meiotic division thereby preventing the extrusion of II Pb (meiotic gynogenesis).

2. By blocking the I cleavage (mitotic gynogenesis)

The degree of homozygosity in meiotic gynogens is supposed to be 50% and mitotic gynogens is 100%. For inducing meiotic gynogenesis shock treatment should be given immediately after fertilization (5 min). For mitotic gynogenesis the shock treatment can be given around 20-30 min after fertilization.

Materials Required

Matured male and female fishes, cotton, ice-blocks, UV irradiation chamber, water bath, thermometer, microscope.

Procedure

1. Inject the required spawning hormones to the brood fishes.

2. Strip the milt freshly from male fish (preferably from another species) an hour before the expected ovulation time of the female fish (the advantage of using milt from different species is that it will be easier to distinguish the gynogens from that of hybrid. The eggs which are fertilized by an unaffected sperm by UV irradiation will develop into a hybrid).

3. Mix the milt with freshly prepared Hank’s solution. Divide the milt into two parts.

4. Keep one half as control and other half for irradiation in a petridish to a 1-2 mm thick.

5. Irradiate stored or freshly collected milt with UV-rays (254 nm, power 1.0 mw/cm at the surface of the milt) under a germicidal lamp for 15-20 min.

6. Check the motility of the sperm after irradiation for different period.

7. After irradiation store the milt in cold temperature or in ice.

8. Strip matured eggs as soon as ovulation commences.

9. Fertilize the freshly stripped eggs with normal and UV irradiated sperm separately.

10. Subject the fertilized eggs to heat, cold shock to inhibit the release of II PB or I cleavage.

11. Incubate the fertilized eggs artificially.

12. Rear the hatched larvae and analyse the chromosome number.

13. Calculate the relative survival rates of the gynogenetic treatment groups and control groups (Survival of gynogenetic group / survival of control group). Monitor the survival at the eye stage, hatching, initiation of feeding and maturation.

14. Compare the growth of the gynogenetic fish and normal diploid fish from hatching to maturity.