شیلات ایران

Interspecific androgenesis in Cyprinids

B. Urbanyi’*, I. Magyary’, M. BercsCnyi2, L. Orb&r3 and L. Horvath’

’ University of Agricultural Sciences, Institute of Animal Husbandry, Biotechnological

Laboratory, Pa’ter K.St. l., H-21 03 Giidiill8, Hungary

2 Warm Water Fish Hatchery Ltd., Szdzhalombatta P$28., H-2441 Hungary

3Agricultural Biotechnology Center, Institute for Molecular Genetics, H-21 01 Giidiill6,

Hungary

*Corresponding author

SUMMARY

Due to increasing demand for preservation of fish genotypes methods suitable for

conservation and restoration of precious, rare or endangered fish species, landraces and

stocks should be developed. Since the long-term preservation of fish ova is not yet possible

(unlike the cryopreservation of fish sperm) the only way to conserve and restore a given

genotype is the combination of sperm cryopreservation and androgenesis. If the ova of a rare

genotype are not available interspecific androgenesis could be applied in order to increase the

efficiency of this combined method.

Viable diploid goldfish (Carassius auratus) progenies were produced through interspecific

androgenesis using common carp (Cyprinus carpio) eggs. Irradiated ova of common carp

(gamma-rays, 25 kR) were fertilized with fresh or cryopreserved goldfish sperm. Diploidy

was restored by application of heat shock (4O”C, 2 min). Colour and morphological markers

were used to prove the male origin of androgenetic progenies. The analyse of genome in the

progeny was carried out with RAPD (Random Amplified Polymorphic DNA) method. Of

twenty-two examined RAPD primary secvences we managed to choose 14, which showed

different pattern of the DNA from the two parents. These experiments proved that the

genome of the progeny was indeed from the goldfish male, but maternal stripes were detected

as well, which were probably of mitochondrial origin.

High fertilization rates and hatching rates were achieved using fresh or frozen goldfish sperm.

Manifestation of phenotypic markers characteristic to common carp was not found among

androgenetic goldfish progenies during half year of development.

Keywords: carp, goldfish, interspecific androgenesis, sperm cryopreservation, genotype

restoration

INTRODUCTION

Fish are ideal objects for genome manipulation: various forms of these techniques are applied

routinely in the daily practice of aquaculture. Androgenesis is a single-parent type of

inheritance where by definition the genetic material of the progeny originates exclusively

from the male gametes (Purdom, 1995), while the maternal chromosomes are presumed to be

totally excluded from the process. In this publication we demonstrate and verify by DNA

analysis, that it is possible to perform interspecific androgenesis between two Cyprinid

species.

MATERIALS AND METHODS

Eggs obtained from a common carp were irradiated by gamma-ray (Purdom, 1995) for the

inactivation of maternal chromosomes and they were then fertilized using cryopreserved

(Magyary et al. 1996a,b) (l-3 weeks prior to propagation) and fresh sperm of goldfish. The

dosage of irradiation was 300 Gray. In controls eggs were not irradiated. Since haploid

embryos are not viable genome of the male pronucleus was duplicated applying heat-shock

on the eggs prior to the first mitotic cleavage. Heat shock parameters (Sheerer et al., 1991)

applied: 2 minutes duration, 40 OC shock temperature starting the shock 37 minutes

postfertilization at 22 OC ambient temperature. Fertilized and shocked eggs were incubated in

Zug jars. Ten to fifteen thousand eggs were used per treatment what made a pilot plant size.

Following a heat shock performed for duplication of the haploid paternal chromosome set,

androgenic goldfish embryos, possessing all three phenotypic recessive markers (triangle tail,

bubble eye, coloration) from their father, but none of the dominant alleles of these genes from

carp, have hatched out (Table 1.).

RED CAP BUBBLE EYE BLACK TELESCOPE

red spot on head bubble eyes telescope eyes

triangle tail triangle tail triangle tail

no barbels no barbels no barbels

I white body red colour black colour

Table 1. Recessive phenotypic markers of the three varieties of goldfish males used for

interspecific androgenesis

Concerning these markers no maternal characters of the carp were detected in androgenetic

groups while the hybrids showed full maternal dominance having single tail, simple eyes and

gray color.

Though the morphological markers gave excellent evidence of the lack of female-derived

intact genes, we have also decided to study the genetic material of the androgenetic

individuals by DNA analysis. Since despite of ongoing research on the isolation of

microsatellite markers from common carp no microsatellites applicable to the investigation of

farmed cyprinids are published or available commercially to date, random amplified

polymorphism of DNA (RAPD) appeared to be the most efficient means of studying the

genetic composition of the androgenetic fry. The oligonucleotide primers were selected on

the basis of the pattern produced with zebrafish DNA, the closest relative of carp and

goldfish thoroughly studied with RAPD analysis.

RESULTS AND DISCUSSION

Out of the twenty two RAPD markers tested eight produced a band pattern different in the

two parents. By testing fourteen androgenic individuals belonging to six families, complete

transmission of all paternal bands could be observed, as demonstrated on one family with two

androgenic individuals tested. DNA analysis allows for studying the fine details of genome

manipulations, which could not be revealed by using traditional techniques. RAPD analysis

appears to be superior to fingerprinting for this purpose, due to its lower template and time

requirement and to its better reproducibility.

In the best batch 28 % of the interspecific androgenetic progenies reached hatching stage

(Figure 1.) and 15 % survived until 2 weeks. No significant differences (~~0.01) were found

between the hatching rates of androgenetic goldfish derived from fresh or frozen sperm. Ten

to thirty androgenetic goldfish individuals per male are being grown up to sexual maturity for

future experiments.

etlilization rate

Figure 1.: Comparison of fertilization and hatching rates of three varieties of androgenic

goldfish derived from irradiated carp eggs using cryopreserved spem. Columns in the first

line indicate hatching rates, while those in the second line the fertilization rates (in percentage

of the total number of eggs fertilized per group for both). The columns mark the average

values of three experiments, the value of which is also shown in numerical form at their top.

It remains a very interesting question to be solved, how could a goldfish embryo with a rather

strict genetic programming grow around a yolk sac considerably larger, than its own.

Parallel application of interspecific androgenesis together with cryopreservation of fish sperm

could be used for restoring species in danger of extinction in the future.

REFERENCES

Purdom, C.E. Genetics anQX+z breeding. 206-207 (Chapman & Hall, London, UK, 1995)

Sheerer, P.D., Thorgard, G.H. & Allendorf, F.W. 1991. J.Exp. Zd. 260,382-390 (1991)

Magyary, I., Urbanyi, B. & Horvath, L. Cryopreservation of common carp (Cyprinus carpio

L.) sperm I. The importance of oxygen supply. Journal of Applied Ichthyology, 12 , 113-l 15,

1996a.

Magyary, I., Urbanyi, B. & Horvath, L. Cryopreservation of common carp (Cyprinus carpio

L.) sperm II. Optimal conditions for fertilization. Journal of Applied Ichthyology, 12, 117-

119,1996b.