OPTIMIZING CONDITIONS FOR ANDROGENESIS INDUCTION IN KOI CARP

Maciej Kwiatkowski¹, Dariusz Kucharczyk¹, Andrzej Szczerbowski², Marek J. Łuczyński², Andrzej Mamcarz¹, Marta Jamróz^1
1 Department of Lake and River Fisheries, University of Warmia and Mazury in Olsztyn, Poland
² Inland Fisheries Institute, Olsztyn-Kortowo, Poland

ABSTRACT

In aquaculture, such genome manipulations as androgenesis and mitotic gynogenesis are of special interest because the resulting progeny are completely homozygous and carries chromosomes from only one parental source. Androgenesis has a lot of potential applications, inc1uding rapid establishment of inbred lines, analyzing patterns of inheritance and recovery of valuable strains or endangered wild populations or species from cryopreserved sperm. In present work, the optimum conditions of ovarian fluids, thermal shock and UV irradiation to involve androgenesis in koi carp (Cyprinus carpio L.) was checked. The best ovarian fluid was one described for common carp by Bongers et al. [5]. As a semi-lethal heat thermal shock was chosen 2 min lasted at 40°C. The optimum dose of genetic inactivation of koi carp oocytes using UV radiation was 3840 J m^-2.

Key words: androgenesis, artificial ovarian fluid, chromosome set manipulation, koi carp thermal shock, UV irradiation.

INTRODUCTION

Owing to dynamic development of biological sciences, during last 30 years was observing significant progress in genomic engineering. Methods of lot manipulations have been worked out, which allow duplicating animal genome or reversing their sex. Androgenesis except gynogenesis and cloning make possible classifying and duplicating individuals. Now it is using in aquaculture for example in production of homozygotic organisms and interspecies hybrids and in reconstruction of threatened populations [1,5,9]. Androgenesis consists in obtaining offspring with nucleus genetic material is identical with paternal genomic material. First step to inactivate oocytes nucleus is performed by X-ray, gamma or UV radiation. The last one method is widely used because of its safety for workers. Above of that, X-ray and gamma radiation induce only partial inactivation of oocytes DNA and they might denature yolk egg proteins [10]. Next step of androgenesis consist in associating oocytes with sperm and application thermal shock to stop first mitotic division. Thermal shock is the most frequent1y used procedure [10,13]. In the case of sex determination, for example XY, man and female individuals are obtained, which permits to reconstruct stock spawners but additional possibilities of stocking specifically classified fish can not be over looked. It is highly important in case of common carp and koi carp, especially, if populations are found with the greatest growth performances or resistance of health or indeterminate climate. Often times price of individuals are rather high, because they are obtained by natural reproduction, but time could be shortened for production of new lines or clones. Androgenesis has also a big potential, because cryopreserved semen might be used to produce fish [10]. The aim of the experiment was to optimalize conditions of koi carp androgenesis.

MATERIAL AND METHODS

Broodstock and collection of gametes
Koi carp (number of spawners: 10 males and 30 females) spawners were collected from Goslawice Fish Farm (central Poland). Fish were transported to the hatchery and kept in 1000 L tanks with controlled temperature (18-20°C) and photoperiod (14 hrs light, 10 hrs dark) conditions [11]. Spawners received injections of GnRHa containing pellets (Ovopel) to involve gamete maturation (in season 0.1 peletes per 1 kg, out of season 0.2 pelets per 1 kg) [10].

In each experiment milt was collected from few males. The quality of sperm was expressed as the percentage of motile spermatozoa. Motility was estimated by microscopic (500X) observation of sperm activated with 0.5% NaCI. Samples of sperm with 75-80% (or more) motile spermatozoa were pooled and used for further treatments. For each experiment eggs were stripped from one female 12-20 hours after the last hormone injection.

Testing ovarian fluids
We used four ovarian fluids: rainbow trout natural ovarian fluid and three artificial: one proposed by Billard (4) for rainbow trout, one proposed by Bongers et al. [5] for common carp and 0.9% NaCI solution. The portions of about 150-200 eggs placed to the ovarian fluids for 5, 15, 30 and 60 min, and after these times fertilization took place. The survival rates were compared to two control groups: one – fertilized at the beginning of the experiment and second - 60 min after first fertilization. All experimental groups were in duplicates.

Heat shock conditions

In this experiment four different temperatures: 35, 38, 40 and 42°C and three different exposed times: 1, 2 and 3 min were tested. Fertilized eggs placed on Petri dishes, 30 min after insemination were moved to the heat bath. After shock eggs were back to the incubation water at 20°C. The survival rates from treated groups were compared to the control one. All experimental groups were in duplicates.

Genetic inactivation of oocytes
Eggs during irradiation were kept on Petri dishes in artificial ovarian fluid composed for common carp [5]. The dishes with eggs were placed on a rocking table with a cyc1e of ~ 1 s. During stirring the eggs were able to roll in the fluid. The UV lamp (30 W, 6.4 W m^-2) was switched on for at least 30 min before the onset of irradiation. Before irradiation of oocytes, control samples of eggs (100-150 eggs in each sample) were fertilized with small volume (0.05 ml) of sperm (control of egg quality: groups K1 and K2). Non-irradiated eggs treated in ovarian fluid were also fertilized using the same volume of milt (control of ovarian fluid quality: groups DlA and DlB). Experimental groups of eggs were fertilized with 0.05 ml of sperm after exposition to the different time of UV irradiation: from 1 to 15 min (dose of UV irradiation ranged from 384 to 5760 J m^-2).

After end of irradiation, eggs from additional control groups were fertilized (control of treatment in ovarian fluid: groups D2A and D2B). The whole procedure was carried out in darkness to avoid genetic photo-reactivation [8]. Eggs were incubated in a laboratory recirculated system at 20°C, which was found as an optimal temperature for carp embryonic development. All experimental groups were repeated.

lncubation
All Petri dishes with eggs from all groups were incubated in aerated aquaria incubated at 20°C.

Statistical analysis
Survival of eggs was calculated as the percentage of the hatched embryos and as a percentage of embryos that started swimming. Differences in hatching success and in survival of koi carp embryos were tested by Duncan’s multiple range test (P < 0.05).

RESULTS

Ovarian fluids
Four different ovarian fluids were tested. The highest survival ratio was observed when the carp eggs placed in fluid proposed by Bongers at al. [5] (Fig.1). High survival rate was noted after 5 and 15 minutes of treatment. That solution was prepared especially for common carp. Results obtained in “Billard” solution were only moderately worse. In other two tested ovarian fluid, the survival was very low (between 0 and 1%), independent of exposition time. When eggs were kept longer in this ovarian fluid ore other fluids, rapid decreasing of survival rates in all experimental groups were observed. After analyzing the results, ovarian fluid proposed by Bongers et al. [5] was chosen for further treatment.

Fig. 1. The results of koi carp egg survival after exposition of two best ovarian fluids: proposed by Billard [4] – “Bi” and Bongers et al. [5] – “Bo”

Thermal shock
Four various temperatures (35, 38, 40 and 42°C) and three variants of thermal shock latency (1, 2, 3 minutes for each thermal shock) were tested. As a semi-lethal shock 40°C for 2 min was chosen (Fig. 2). At highest temperature tested: 42°C very high mortalities are observed.

Fig. 2. The results of testing different parameters of heat shock on koi carp eggs

UV irradiation
The highest ratios of survival to eyed-egg-stage and free-embryo-stage of haploids were observed after 10 minutes exposure on UV radiation (3840 Jm^-2) (Fig. 3). All hatched embryos were characteristically deformed, such as shown in Photo 4. The lowest survival rate was found after of 15 minutes irradiation. In sites of 1 and 3 minutes at exposure hatching of diploid (larvae without visible abnormalities) fish was observed.

Fig. 3. The influence of UV irradiation on koi carp embryos survival

Photo 4. Koi carp haploids (phot. R. Kujawa)

DISCUSSION

Rapid development of aquaculture during recent years is connected with development of different biotechnological methods. One of these methods, which may be used for example to produce fish c1ones, is androgenesis. This complicated method enquires a lot of primary work to optimalize all conditions of this process [10]. During genetic inactivation DNA of oocytes, the eggs of cyprinids should be placed in ovarian fluids to avoid of drying [5]. For this reason in koi carp different natural or artificial ovarian fluids were tested. In the present study, the artificial ovarian fluid composed for common carp by Bongers et al. [5] worked the best in koi Carp. Similar results were obtained for eggs of common bream (Abramis brama L.) [10]. Quite good results in this study were also obtained by testing artificial ovarian fluid proposed for rainbow trout by Billard [4]. This last fluid worked well in other cyprinid, like ide (Leuciscus idus L.) or dace (Leuciscus leuciscus L.) [9,10]. High survival rate observed during first 15 min of eggs treatment in ovarian fluid is enough to obtain genetic inactivation of oocytes. Environmental shock in the case of androgenesis is applied to block first mitotic division in the zygote. There are few kinds of shock, which might be applied: thermal (cold or heat), pressure, chemical or electric [7,14,16,17]. When androgenesis is used in cyprinids, usually thermal shock is applied. For warm-water species, such as common carp, common bream or common tench (Tinca tinca L.) usually temperature shock at 40-42°C; for cold-water: ide and dace 35-38°C. Other kind of shock: cold-temperature or pressure was usually much less effective than heat-temperature shock [10]. In order to induce genetic inactivation of koi carp oocytes the UV radiation was chosen. This kind of ionising radiation was earlier successfully applied in loach, cyprinids, sturgeons and cichlids [2,3,6,12]. The optimal UV irradiation doses were different and dependent of oocytes size and kind of ovarian liquid, but in all cases ranged between 2000 and 10 000 Jm^-2. In case of koi carp, the optimal dose was about 4000 Jm^-2 and was quite similar to other cyprinids: 3500 Jm^-2 for common tench and 4600 Jm^-2 for ide and dace [10]. These irradiation doses were very high and were about 10-times higher than those used for water sterilization of strong bacteria such as Thiobacillus intermedis or Halobacterium salinarum [15]. If genetic inactivation of oocytes was well performed haploid organisms were developed. Haploids of cyprinids normally developed up to eyed egg-stage and died. Only a limited number of such embryos hatched. All haploid embryos usually showed typical body deformations.

CONCLUSIONS

1. Highest embryos survival was noted when artificial ovarian fluid was used for keeping eggs during UV-irradiation.

2. Thermal shock describes as 40°C and duration 2 min was semi-lethal for carp eggs.

3. The optimum UV irradiation dose for carp eggs was 3840 Jm^-2.

REFERENCES

1. Arai K., Masaoka T., Suzuki R., 1992. Optimum conditions of UV ray irradiation for genetic inactivation of loach eggs. Nippon Suisan Gakkaishi 58, 1197-1201.

2. Arai, K., Ikeno M., Suzuki R., 1995. Production of androgenic diploid loach Misgurnus anguillicaudatus using spermatozoa of natural tetraploids. Aquaculture 137, 131-138.

3. Bercsenyi M., Magyary I., Urbanyi B., Orban L., Horvath L., 1998. Hatching out goldfish from common carp eggs: interspecyfic androgenesis between two cyprinid fishes. Genome 41, 573-579.

4. Billard R., 1974. Artificial insemination of the trout, Salmo gairdneri Richardson. IV-Effects of K⁺ and Na⁺ ions on gamete fertilizing ability. Bull. Fr. Piscic. 256, 88-100.

5. Bongers A.B.J., Veld E.P.C., Abo-Hasema K., Bremmer I.M., Eding E.H., Komen J., Richter C.J.J., 1994. Androgenesis in common carp (Cyprinus carpio L.) using UV irradiation in a synthetic ovarian fluid and heat shock. Aquaculture 122, 119-132.

6. Grunina A.S., Recubratsky A.V., Emelyanowa O.V., Neyfakh A.A., 1995. Induced androgenesis in fish: Production of viable androgenetic diploid hybrids. Aquaculture 137, 149.

7. Hussain M.G., McAndrew B.J., Penman D.J., Sodusk P., 1994. Estimate gene centromer recombination frequencies in diploids of Oreochromis niloticus (L), using allozymes, skin colour and a putative sex determination lows (SDL-2). In A.R. Beamont (ed.): Genetics and Evolution of Aquatic organisms. Chapman and Hall, London, UK, 502-509.

8. Kaastrup P., Horlyck V., 1987. Development of a simple method to optimize the conditions of producing gynogenetic offspring, Using albino rainbow trout, Salmo gairdneri Richardson, females as an indicator for gynogenesis. J. Fish Biol. 31, 29-33.

9. Kucharczyk D., 2001. Genetic inactivation of Leuciscus idus L. (ide) oocytes using UV irradiation. Cytobios 104, 189-195.

10. Kucharczyk D., 2002. Rozród kontrolowany i androgeneza wybranych gatunków ryb karpiowatych [Artificial reproduction and androgenesis of some cyprinids]. Rozpr. i monogr. Wydaw. UWM, Olsztyn 63, 1-80 [in Polish].

11. Kujawa R., Kucharczyk D., Mamcarz A., 1999. A model system for keeping spawners of wild and domestic fish before artificial spawning. Aquacult. Engineering 20, 85-89.

12. Marengoni N.G, Onoue Y., 1998. Ultraviolet induced androgenesis in Nile tlapia, Oreochromis niloticus (L.), and hybrid Nile X blue tilapia, O. aureus (Steindachner). Aquacult. Res. 29, 359-366.

13. Pandian T.J., Koteeswaran R., 1998. Ploidy level and sex control in fish. Hydrobiologia 384, 167-243.

14. Peruzzi S., Chatain B., 2000. Pressure and cold shock induction of meiotic gynogenesis and triploidy in the European see bass, Dicentrachus labrax L.: relative efficiency of methods and paternal variability. Aquaculture 189, 23-37.

15. Shahmohammadi H.R., Asgarani E., Terato H., Ide H., Yamamoto O., 1997. Effects of super (60) gamma-rays, ultraviolet light, and mitocin c on Halobacterium salinarum nad Thiobacillus intermedius. J. Radiat. Res. 38, 37-43.

16. Siraj S.S., Seki S., Jee A.K., Yamada Y., 1993. Diploid gynogenesis in Pampan Jawa Puntius gonionotus using UV irradiated sperm of Puntius Schwanenfeldii. Nippon Suisan Gakkaisshi 59, 957-962.

17. Varadaraj K., Pandian T.J., 1990. Production of all-female sterile-triploid Oreochromis mossambicus. Aquaculture 84, 117-123.

 

Accepted for print: 12.03.2008

---

Maciej Kwiatkowski
Department of Lake and River Fisheries,
University of Warmia and Mazury in Olsztyn, Poland
Oczapowskiego 5, 10-718 Olsztyn-Kortowo, Poland
email: thornghost@.onet.pl

Dariusz Kucharczyk
Department of Lake and River Fisheries,
University of Warmia and Mazury in Olsztyn, Poland
M. Oczapowskiego 5, 10-718 Olsztyn-Kortowo, Poland

Andrzej Szczerbowski
Inland Fisheries Institute,
Olsztyn-Kortowo, Poland
Oczapowskiego 10, 10-719 Olsztyn-Kortowo, Poland

Marek J. Łuczyński
Inland Fisheries Institute,
Olsztyn-Kortowo, Poland
Oczapowskiego 10, 10-719 Olsztyn-Kortowo, Poland

Andrzej Mamcarz
Department of Lake and River Fisheries,
University of Warmia and Mazury in Olsztyn, Poland
M. Oczapowskiego 5, 10-957 Olsztyn, Poland
phone +48 89 523 33 88,
fax +48 89 523 39 69
email: mamcarz@uwm.edu.pl

Marta Jamróz
Department of Lake and River Fisheries,
University of Warmia and Mazury in Olsztyn, Poland
Oczapowskiego 5, 10-718 Olsztyn-Kortowo, Poland

---

Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed 'Discussions' and hyperlinked to the article.

---

Main  -  Issues  -  How to Submit  -  From the Publisher  -  Search  -  Subscription