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Induction of meiotic gynogenesis in sterlet (Acipenser ruthenus) using UV-irradiated bester sperm
Aquaculture 264 (2007) 54 – 58 www.elsevier.com/locate/aqua-online
Induction of meiotic gynogenesis in sterlet (Acipenser ruthenus) using UV-irradiated bester sperm Dorota Fopp-Bayat a,⁎, Ryszard Kolman b , Pawel Woznicki a a
Department of Ichthyology, Faculty of Environmental Sciences and Fisheries, University of Warmia and Mazury in Olsztyn, Ul. Oczapowskiego 5, 10-718 Olsztyn-Kortowo, Poland b Department of Icthyology, Inland Fisheries Institute in Olsztyn, Ul. Oczapowskiego 10, 10-718 Olsztyn-Kortowo, Poland Received 28 July 2006; received in revised form 30 November 2006; accepted 1 December 2006
Abstract Diploid gynogenesis was induced in sterlet (Acipenser ruthenus) using UV-irradiated bester (Huso huso × A. ruthenus) sperm. The optimal condition for the retention of the second polar body in sterlet was investigated by altering the timing, intensity and duration of heat shock application. A total of 90 gynogens of known parentage from three different experimental treatments were screened using microsatellite DNA analysis, and uniparental transmission in meiogens was confirmed. © 2007 Elsevier B.V. All rights reserved. Keywords: Acipenser ruthenus; Bester; Gynogenesis; Microsatellite DNA; Sterlet; UV-irradiation
1. Introduction Sturgeons are a very ancient fish group, existing since the Late Cretaceous with a wide distribution in the Northern Hemisphere (Grande and Bemis, 1991). Sturgeon biology is interesting because of important conservation and economic issues involving these fishes. Sturgeons are the source of two high-value products: boneless and very tasteful meat and black caviar. Fish farms producing sturgeon meat and caviar as well as reproducing sturgeons under controlled conditions have emerged (Wade and Fadel, 1997; Chebanov and Billard, 2001). Sturgeons reared under controlled conditions show rapid growth and reach
⁎ Corresponding author. Tel.: +48 89 5234772; fax: +48 89 5233754. E-mail address: [email protected] (D. Fopp-Bayat). 0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.12.006
sexual maturity faster than in nature (Chebanov and Billard, 2001). Sterlet (Acipenser ruthenus) is the species with relatively small size and rapid sexual maturation within the family Acipenseridae (Nikolskij, 1971; Sokolov and Vasiliev, 1989). These life history traits make the sterlet a useful model for the genome manipulations in Acipenseridae. Gynogenesis is a developmental manipulation facilitating the inheritance of maternal genetic material alone, which has been accomplished in several fish species (Ihssen et al., 1990; Pandian and Koteeswaran, 1998; Arai, 2001; Paschos et al., 2001). This genome manipulation technique involves the activation of egg development by genetically inactive spermatozoa and diploidy restoration by retention of the second polar body. The first experiments on induced gynogenesis in sturgeons were reported by Romashov et al. (1963). The next studies on techniques for production of meiotic gynogenetic sturgeons were conducted on the American
D. Fopp-Bayat et al. / Aquaculture 264 (2007) 54–58
55
acipenserids and polyodontid: Acipenser transmontanus (Van Eenennaam et al., 1996), Polyodon spathula (Mims et al., 1997) and Scaphirhynchus platorynchus (Mims and Shelton, 1998). Recoubratsky et al. (2003) successfully induced meiotic gynogenesis in Acipenser gueldenstaedti and Acipenser stellatus, but in A. ruthenus they failed. The objective of the present study was to induce meiotic gynogenesis in sterlet and to confirm the success of this gynogenesis using microsatellite DNA markers.
to spawn by two injections of 6 mg kg− 1 body weight of Abramis brama pituitary extracts and was transferred to a recirculating water system at 15 °C (isolated from the male). One male bester was induced to spermiate by a single injection of 1 g kg− 1 body weight of Ovopel (mammalian GnRH analogue + metoclopramide). Ovulated oocytes were obtained by stripping the female 20– 24 h following hormonal injection. Sperm was drawn from the male into a syringe and kept at 4 °C before use. Sperm motility was checked under light microscopy after activation with freshwater.
2. Material and methods
2.2. Sperm and ova treatment
2.1. Experimental design and gamete collection
Irradiation was carried out using different exposure times (45 s, 60 s and 70 s). For UV-irradiation, 2 ml of sperm was diluted with 18 ml of seminal fluid (supernatant from surplus semen centrifuged at 8000 rpm for 15 min) and put into Petri dishes (diameter: 100 mm) to a depth of approximately 1 mm. These dishes were placed on a gently rotating platform (90 rpm) 50 cm below the UV lamp (Philips 15 W). Sperm was treated with UV-irradiation for 45, 60 or 70 s. After irradiation, 20 ml of 15 °C water from the incubation system was added to the irradiated sperm suspension, and this mixture was immediately added to ova. Eggs were divided into nine approximately equal groups (∼ 1000 eggs), held in individual 2 l beakers and fertilized with 3.25 ml of diluted irradiated or normal sperm at 15 °C. Egg batches were fertilized with control sperm prediluted in seminal fluid or with irradiated spermatozoa, as described above. Three batches of eggs used as a haploid control, activated with UV-irradiated sperm, were not heat-shocked. Eggs to be heat-shocked were transferred into boxes with perforated mesh and kept in water at 34 °C for 2 min. Heat shocks were applied at 18 min after fertilization in experiments by soaking the boxes in a polystyrene incubator containing heated water at 34 °C. The temperature in the incubator was constantly monitored. Immediately after treatment, eggs were transferred into 1000 ml Weiss incubators with their controls in a thermoregulated incubation system at 15 °C. Additionally, ova quality was checked by insemination with sterlet milt (Table 1). Survival of developing eggs and viable fry was recorded at different developmental stages: fertilization (2 h after fertilization—a.f.), neurulation (50–60 h a.f.) and hatching (6 days a.f.). Percent of neurulation was determined by observation of the neural tube closure at 50–60 h after fertilization (Dettlaff, 1993). At 6–8 days post-fertilization, the number of normal, free-swimming larvae was counted for each treatment and percent survival
The experiment was conducted at the Wasosze fish farm near Konin (Poland) in the winter of 2006 using sterlet oocytes and bester spermatozoa. The experimental design is presented in Table 1. The experiment included a diploid control group, a haploid control group to determine the efficiency of UV inactivation of the sperm (untreated eggs and UV-irradiated sperm), a triploid control group to determine the efficacy of second polar body retention (temperature-shocked eggs and untreated sperm), and a treatment designed to induce gynogenesis (temperature-shocked eggs and UV-irradiated sperm). Maturation in females was assessed by measuring the diameter of oocytes and observing germinal vesicle migration in ovarian biopsies. One female was induced Table 1 Experimental design and survival rates at different developmental stages of larvae for meiotic gynogenesis in sterlet Design
Egg treatment
Sperm treatment a/ Gast. species (%)
Neurul. (%)
Hatch (%)
Diploid
Untreated
88
65
53
Diploid
Untreated
Untreated/ sterlet Untreated/ bester 45 s/bester 60 s/bester 70 s/bester Untreated/ bester 45 s/bester
74
54
42
36 33 30 73
30 21 24 53
20 13 10 41
35
28
24
60 s/bester
35
29
25
70 s/bester
28
22
19
Haploid Haploid Haploid Triploid
Untreated Untreated Untreated 34 °C– 2 min Gynogen 34 °C– 2 min Gynogen 34 °C– 2 min Gynogen 34 °C– 2 min a
Sperm treatments involved UV-irradiation for the number of seconds indicated.
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D. Fopp-Bayat et al. / Aquaculture 264 (2007) 54–58
was noted. Hatched larvae from each individual treatment were transferred to a special system of tanks (10 l each) at a temperature of 17 °C, where they remained until sampling for ploidy and microsatellite DNA analysis. The survival was recorded daily from 1 day post-hatch. Larval feeding was initiated at 6 days post-hatch. 2.3. Genetic investigation of gynogenesis effect Thirty randomly selected larvae from putative gynogenetic groups and controls were sampled at 2–6 days after hatching and stored in 96% ethanol. Fin-clips from 2 parental individuals: female sterlet, and male bester (hybrid of beluga and sterlet) were stored in 96% ethanol. Genomic DNA for amplification of three microsatellite loci [Afu-68, Afu-19 (May et al., 1997) and Aox-45 (King et al., 2001)] was extracted using Chelex 100 (Walsh et al., 1991). Reaction mixes were prepared in a total volume of 25 μl with 40 ng DNA template, 1× PCR reaction buffer (50 mM KCl, pH 8.5; Triton X-100), 0.4 mM of each primer, 0.25 mM of each deoxynucleotide triphosphate (dNTP), 3.3 mM MgCl2 and 0.6 U Go Taq Flexi DNA Polymerase (Promega, Madison, WI, USA). Re-distilled water was used to bring the reaction mixture to the desired final volume. Amplification was conducted with a Mastercycler gradient thermocycler (Eppendorf, Germany), with initial denaturation at 94 °C for 5 min, followed by 33 amplification cycles (94 °C, 1 min; 53–55 °C, 30 s; 72 °C–30 s), and final elongation at 72 °C for 5 min. Aliquots containing PCR products and reaction buffer were electrophoresed using a 6% polyacrylamide gel, and DNA bands were visualized by the silver staining method (Tegelström, 1986). Electrophoresis was conducted on a Bio-Rad SequiGen Sequencing Cellsystem, and the gel size was 38 × 30 cm. Amplified fragments were sized by comparing migration with two DNA standards: ϕX 174 DNA/Hinf I DNA Step Ladder (Promega, Madison, WI, USA) and 25 bp DNA Step Ladder (Promega, Madison, WI, USA). Every gel analyzing progeny included two lanes containing the appropriate parental microsatellite PCR amplification products. Specific microsatellite profiles for parents were noted and compared to those from experimental groups. Ten specimens from each gynogenetic group were analyzed cytogenetically. Chromosomes were prepared according to Woznicki et al. (1998) with modifications. At least 5 wellspread metaphase plates from each specimen were analyzed.
3. Results The survival to hatching of fish in putative gynogenetic and haploid treatments varied considerably with the duration of UV exposition. Fig. 1A and B shows the survival of hatched larvae in gynogenetic and haploid groups compared to the control group. UVirradiation of 60 s and 70 s was found to be optimal for producing viable putative gynogenetic diploids. A duration of 2 min was optimal when eggs were heatshocked at 34 °C starting in the 18th minute after fertilization to retain the second polar body and to produce gynogenetic diploids. Most of the embryos in the haploid control groups died at an early stage of development before neurulation, or they displayed features of “haploid syndrome” (abnormal body shape, open blastopore) and failed to hatch normally. Unlike haploid larvae, the obtained gynogens have started to feed, but after a few days the water condition drastically decreased and this fact was the cause of gynogens lost. The results of microsatellite DNA analysis showed that there was no genetic contribution from the paternal
Fig. 1. Survival of gynogenetic, haploid and control groups of hybrid sterlet × bester. A — haplo 1, haplo 2, haplo 3 — sperm inactivation through 45 s, 60 s and 70 s respectively; B — gyno 1, gyno 2, gyno 3 — sperm inactivation through 45 s, 60 s and 70 s respectively; x-axis: days post-hatch.
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Fig. 2. Afu-68 allele segregation in gynogenetic offspring of sterlet (Acipenser ruthenus). M1 — marker 25 bp (Promega), M2 — marker ϕX 174 (Promega), gynogenetic offspring — samples: 1–13, female of sterlet — sample 14, male of beluga × sterlet — sample 15.
genome in the gynogenetic group. Locus Afu-68 was monomorphic in studied groups of gynogenetic and haploid fish, and an allele 204 base pairs (bp) was observed in those groups of fish (Fig. 2). The allele 204 bp was characteristic for the mother of the gynogenetic offspring, while the male (sperm donor) exhibited alleles of 230 and 136 bp. Loci Afu-19 and
Aox-45 were polymorphic in studied gynogenetic groups of sterlet, because of the heterozygous state of the gynogenotes' mother. At locus Afu-19, two alleles of 138 and 141 bp were observed in diploid gynogens of sterlet, while at locus Aox-45, alleles of 133 and 145 bp were observed. The sperm donor possessed two alleles of locus Afu-19 (165 and 138 bp) and one allele of locus Aox-45 (121 bp). The cytogenetic analysis showed that the chromosome number in all studied gynogenetic individuals was diploid (∼ 120 chromosomes in metaphase plates) (Fig. 3). 4. Discussion
Fig. 3. Metaphase chromosome set from diploid gynogenetic sterlet (Acipenser ruthenus).
Induction of gynogenesis and polyploidy in sturgeon species is of interest to commercial aquaculturists and to researchers investigating genetics and the mechanism of sex determination in fishes. In sturgeons, the induction of gynogenesis would be important for potential production of all-female populations for caviar production. In the present research, the production of gynogenetic sterlet was described. The haploid embryos displayed the same “haploid syndrome” as was
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described by Van Eenennaam et al. (1996) in white sturgeon. Irradiation shock duration of 60 s and 70 s was optimal for bester sperm that was used for fertilization of eggs of sterlet. The UV-irradiation to deactivate the male genome prior to gynogenesis created some difficulties due to the small size of bester spermatozoa with acrosomes. The spermatozoa of the sterlet (and probably bester, too) are more sensitive to UV exposure than spermatozoa of the other sturgeons (Recoubratsky et al., 2003). However, the failure of sterlet gynogenesis in the study of Recoubratsky et al. (2003) was probably caused by too strict conditions for heat shock of the eggs (37 °C for 2.5 min in comparison to 34 °C for 2 min in the present study). A method to provide genetic verification of gynogenesis in progeny was shown. Microsatellite DNA analysis revealed that individuals from haploid (haplo 2 and haplo 3) and gynogenetic (gyno 2 and gyno 3) groups had no apparent paternal inheritance. The results of this study may be useful for future research. Irradiated bester (and probably sterlet) sperm could be useful for gynogenesis induction in other sturgeon species (as a heterologous sperm). Acknowledgments The study was supported by the University of Olsztyn grant no. 080302.0205. We thank Ms. Elzbieta Fopp and Mr. Andrzej Fopp from the Wasosze fish farm for kindly providing fish for the study. We also thank the anonymous reviewers for useful comments on the manuscript. References Arai, K., 2001. Genetic improvement of aquaculture finfish species by chromosome manipulation techniques in Japan. Aquaculture 197, 205–228. Chebanov, M., Billard, R., 2001. The culture of sturgeons in Russia: production of juveniles for stocking and meat for human consumption. Aquat. Living Resour. 14, 375–381. Dettlaff, T.A., 1993. Sturgeon fishes. In: Dettlaff, T.A., Ginzburg, A.S., Schmalhausen, O.L. (Eds.), Developmental Biology and Aquaculture. Springer-Verlag, Berlin, pp. 67–71. Grande, L., Bemis, W.E., 1991. Osteology and phylogenetic relationships of fossil and recent paddlefish (Polyodontidae) with comments on interrelationships of Acipenseriformes. J. Vertebr. Paleontol. 11 (suppl. No 1) 121 pp.
Ihssen, P.E., McKay, L.R., McMillan, I., Phillips, R.B., 1990. Ploidy manipulation and gynogenesis in fishes: cytogenetic and fisheries applications. Trans. Am. Fish. Soc. 119, 698–717. King, T.L., Lubinski, B.A., Spidle, A.P., 2001. Microsatellite DNA variation in Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus) and cross-species amplification in the Acipenseridae. Conserv. Genet. 2, 103–119. May, B., Krueger, C.C., Kincaid, H.L., 1997. Genetic variation at microsatellite loci in sturgeon: primer sequence homology in Acipenser and Scaphirhynchus. Can. J. Fish. Aquat. Sci. 54, 1542–1547. Mims, S.D., Shelton, W.L., 1998. Induced meiotic gynogenesis in shovelnose sturgeon. Aquac. Int. 6, 323–329. Mims, S.D., Shelton, W.L., Linhart, O., Wang, C., 1997. Induced meiotic gynogenesis of paddlefish Polyodon spathula. J. World Aquac. Soc. 28, 334–343. Nikolskij, G.V., 1971. Chastnaya ikhtiologiya. Vysshaaya shkola, Moscow, Russia. 471 pp. (in Russian). Pandian, T.J., Koteeswaran, R., 1998. Ploidy induction and sex control in fish. Hydrobiologia 384, 167–243. Paschos, I., Natsis, L., Nathanailides, C., Kagalou, I., Kolettas, E., 2001. Induction of gynogenesis and androgenesis in goldfish Carassius auratus (var. oranda). Reprod. Domest. Anim. 36, 195–198. Recoubratsky, A.V., Grunina, A.S., Barmintsev, V.A., Golovanova, T.S., Chudinov, O.S., Abramova, A.B., Panchenko, N.S., Kupchenko, S.A., 2003. Meiotic gynogenesis in the stellate and Russian sturgeon and sterlet. Russ. J. Dev. Biol. 34, 92–101. Romashov, D.D., Nikolyukin, N.I., Belyaeva, V.N., Timofeeva, N.A., 1963. Possibilities of producing diploid radiation-induced gynogenesis in sturgeons. Radiobiologiya 3, 104–110 (in Russian). Sokolov, L.I., Vasiliev, V.P., 1989. Acipenser ruthenus Linneaus. In: Holcik, J. (Ed.), The Freshwater Fishes of Europe, vol. I/II. AULA-Verlag, Wiesbaden, pp. 227–262. Tegelström, H., 1986. Mitochondrial DNA in natural populations: an improved routine for the screening of genetic variation based on sensitive silver staining. Electrophoresis 7, 226–229. Van Eenennaam, A.L., Van Eenennaam, J.P., Medrano, J.F., Doroshov, S.I., 1996. Rapid verification of meiotic gynogenesis and polyploidy in white sturgeon (Acipenser transmontanus Richardson). Aquaculture 147, 177–189. Wade, E.M., Fadel, J.G., 1997. Optimization of caviar and meat production from white sturgeon (Acipenser transmontanus). Agric. Syst. 54, 1–21. Walsh, P.S., Metzger, D.A., Higuchi, R., 1991. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques 10, 506–513. Woznicki, P., Jankun, M., Luczynski, M., 1998. Chromosome polymorphism in Salmo trutta morpha lacustris from Poland, Wdzydze Lake population: variation in the short arm length of chromosome eleven. Aquat. Sci. 60, 367–375.
Induction of meiotic gynogenesis in sterlet (Acipenser ruthenus) using UV-irradiated bester sperm Dorota Fopp-Bayat a,⁎, Ryszard Kolman b , Pawel Woznicki a a
Department of Ichthyology, Faculty of Environmental Sciences and Fisheries, University of Warmia and Mazury in Olsztyn, Ul. Oczapowskiego 5, 10-718 Olsztyn-Kortowo, Poland b Department of Icthyology, Inland Fisheries Institute in Olsztyn, Ul. Oczapowskiego 10, 10-718 Olsztyn-Kortowo, Poland Received 28 July 2006; received in revised form 30 November 2006; accepted 1 December 2006
Abstract Diploid gynogenesis was induced in sterlet (Acipenser ruthenus) using UV-irradiated bester (Huso huso × A. ruthenus) sperm. The optimal condition for the retention of the second polar body in sterlet was investigated by altering the timing, intensity and duration of heat shock application. A total of 90 gynogens of known parentage from three different experimental treatments were screened using microsatellite DNA analysis, and uniparental transmission in meiogens was confirmed. © 2007 Elsevier B.V. All rights reserved. Keywords: Acipenser ruthenus; Bester; Gynogenesis; Microsatellite DNA; Sterlet; UV-irradiation
1. Introduction Sturgeons are a very ancient fish group, existing since the Late Cretaceous with a wide distribution in the Northern Hemisphere (Grande and Bemis, 1991). Sturgeon biology is interesting because of important conservation and economic issues involving these fishes. Sturgeons are the source of two high-value products: boneless and very tasteful meat and black caviar. Fish farms producing sturgeon meat and caviar as well as reproducing sturgeons under controlled conditions have emerged (Wade and Fadel, 1997; Chebanov and Billard, 2001). Sturgeons reared under controlled conditions show rapid growth and reach
⁎ Corresponding author. Tel.: +48 89 5234772; fax: +48 89 5233754. E-mail address: [email protected] (D. Fopp-Bayat). 0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.12.006
sexual maturity faster than in nature (Chebanov and Billard, 2001). Sterlet (Acipenser ruthenus) is the species with relatively small size and rapid sexual maturation within the family Acipenseridae (Nikolskij, 1971; Sokolov and Vasiliev, 1989). These life history traits make the sterlet a useful model for the genome manipulations in Acipenseridae. Gynogenesis is a developmental manipulation facilitating the inheritance of maternal genetic material alone, which has been accomplished in several fish species (Ihssen et al., 1990; Pandian and Koteeswaran, 1998; Arai, 2001; Paschos et al., 2001). This genome manipulation technique involves the activation of egg development by genetically inactive spermatozoa and diploidy restoration by retention of the second polar body. The first experiments on induced gynogenesis in sturgeons were reported by Romashov et al. (1963). The next studies on techniques for production of meiotic gynogenetic sturgeons were conducted on the American
D. Fopp-Bayat et al. / Aquaculture 264 (2007) 54–58
55
acipenserids and polyodontid: Acipenser transmontanus (Van Eenennaam et al., 1996), Polyodon spathula (Mims et al., 1997) and Scaphirhynchus platorynchus (Mims and Shelton, 1998). Recoubratsky et al. (2003) successfully induced meiotic gynogenesis in Acipenser gueldenstaedti and Acipenser stellatus, but in A. ruthenus they failed. The objective of the present study was to induce meiotic gynogenesis in sterlet and to confirm the success of this gynogenesis using microsatellite DNA markers.
to spawn by two injections of 6 mg kg− 1 body weight of Abramis brama pituitary extracts and was transferred to a recirculating water system at 15 °C (isolated from the male). One male bester was induced to spermiate by a single injection of 1 g kg− 1 body weight of Ovopel (mammalian GnRH analogue + metoclopramide). Ovulated oocytes were obtained by stripping the female 20– 24 h following hormonal injection. Sperm was drawn from the male into a syringe and kept at 4 °C before use. Sperm motility was checked under light microscopy after activation with freshwater.
2. Material and methods
2.2. Sperm and ova treatment
2.1. Experimental design and gamete collection
Irradiation was carried out using different exposure times (45 s, 60 s and 70 s). For UV-irradiation, 2 ml of sperm was diluted with 18 ml of seminal fluid (supernatant from surplus semen centrifuged at 8000 rpm for 15 min) and put into Petri dishes (diameter: 100 mm) to a depth of approximately 1 mm. These dishes were placed on a gently rotating platform (90 rpm) 50 cm below the UV lamp (Philips 15 W). Sperm was treated with UV-irradiation for 45, 60 or 70 s. After irradiation, 20 ml of 15 °C water from the incubation system was added to the irradiated sperm suspension, and this mixture was immediately added to ova. Eggs were divided into nine approximately equal groups (∼ 1000 eggs), held in individual 2 l beakers and fertilized with 3.25 ml of diluted irradiated or normal sperm at 15 °C. Egg batches were fertilized with control sperm prediluted in seminal fluid or with irradiated spermatozoa, as described above. Three batches of eggs used as a haploid control, activated with UV-irradiated sperm, were not heat-shocked. Eggs to be heat-shocked were transferred into boxes with perforated mesh and kept in water at 34 °C for 2 min. Heat shocks were applied at 18 min after fertilization in experiments by soaking the boxes in a polystyrene incubator containing heated water at 34 °C. The temperature in the incubator was constantly monitored. Immediately after treatment, eggs were transferred into 1000 ml Weiss incubators with their controls in a thermoregulated incubation system at 15 °C. Additionally, ova quality was checked by insemination with sterlet milt (Table 1). Survival of developing eggs and viable fry was recorded at different developmental stages: fertilization (2 h after fertilization—a.f.), neurulation (50–60 h a.f.) and hatching (6 days a.f.). Percent of neurulation was determined by observation of the neural tube closure at 50–60 h after fertilization (Dettlaff, 1993). At 6–8 days post-fertilization, the number of normal, free-swimming larvae was counted for each treatment and percent survival
The experiment was conducted at the Wasosze fish farm near Konin (Poland) in the winter of 2006 using sterlet oocytes and bester spermatozoa. The experimental design is presented in Table 1. The experiment included a diploid control group, a haploid control group to determine the efficiency of UV inactivation of the sperm (untreated eggs and UV-irradiated sperm), a triploid control group to determine the efficacy of second polar body retention (temperature-shocked eggs and untreated sperm), and a treatment designed to induce gynogenesis (temperature-shocked eggs and UV-irradiated sperm). Maturation in females was assessed by measuring the diameter of oocytes and observing germinal vesicle migration in ovarian biopsies. One female was induced Table 1 Experimental design and survival rates at different developmental stages of larvae for meiotic gynogenesis in sterlet Design
Egg treatment
Sperm treatment a/ Gast. species (%)
Neurul. (%)
Hatch (%)
Diploid
Untreated
88
65
53
Diploid
Untreated
Untreated/ sterlet Untreated/ bester 45 s/bester 60 s/bester 70 s/bester Untreated/ bester 45 s/bester
74
54
42
36 33 30 73
30 21 24 53
20 13 10 41
35
28
24
60 s/bester
35
29
25
70 s/bester
28
22
19
Haploid Haploid Haploid Triploid
Untreated Untreated Untreated 34 °C– 2 min Gynogen 34 °C– 2 min Gynogen 34 °C– 2 min Gynogen 34 °C– 2 min a
Sperm treatments involved UV-irradiation for the number of seconds indicated.
56
D. Fopp-Bayat et al. / Aquaculture 264 (2007) 54–58
was noted. Hatched larvae from each individual treatment were transferred to a special system of tanks (10 l each) at a temperature of 17 °C, where they remained until sampling for ploidy and microsatellite DNA analysis. The survival was recorded daily from 1 day post-hatch. Larval feeding was initiated at 6 days post-hatch. 2.3. Genetic investigation of gynogenesis effect Thirty randomly selected larvae from putative gynogenetic groups and controls were sampled at 2–6 days after hatching and stored in 96% ethanol. Fin-clips from 2 parental individuals: female sterlet, and male bester (hybrid of beluga and sterlet) were stored in 96% ethanol. Genomic DNA for amplification of three microsatellite loci [Afu-68, Afu-19 (May et al., 1997) and Aox-45 (King et al., 2001)] was extracted using Chelex 100 (Walsh et al., 1991). Reaction mixes were prepared in a total volume of 25 μl with 40 ng DNA template, 1× PCR reaction buffer (50 mM KCl, pH 8.5; Triton X-100), 0.4 mM of each primer, 0.25 mM of each deoxynucleotide triphosphate (dNTP), 3.3 mM MgCl2 and 0.6 U Go Taq Flexi DNA Polymerase (Promega, Madison, WI, USA). Re-distilled water was used to bring the reaction mixture to the desired final volume. Amplification was conducted with a Mastercycler gradient thermocycler (Eppendorf, Germany), with initial denaturation at 94 °C for 5 min, followed by 33 amplification cycles (94 °C, 1 min; 53–55 °C, 30 s; 72 °C–30 s), and final elongation at 72 °C for 5 min. Aliquots containing PCR products and reaction buffer were electrophoresed using a 6% polyacrylamide gel, and DNA bands were visualized by the silver staining method (Tegelström, 1986). Electrophoresis was conducted on a Bio-Rad SequiGen Sequencing Cellsystem, and the gel size was 38 × 30 cm. Amplified fragments were sized by comparing migration with two DNA standards: ϕX 174 DNA/Hinf I DNA Step Ladder (Promega, Madison, WI, USA) and 25 bp DNA Step Ladder (Promega, Madison, WI, USA). Every gel analyzing progeny included two lanes containing the appropriate parental microsatellite PCR amplification products. Specific microsatellite profiles for parents were noted and compared to those from experimental groups. Ten specimens from each gynogenetic group were analyzed cytogenetically. Chromosomes were prepared according to Woznicki et al. (1998) with modifications. At least 5 wellspread metaphase plates from each specimen were analyzed.
3. Results The survival to hatching of fish in putative gynogenetic and haploid treatments varied considerably with the duration of UV exposition. Fig. 1A and B shows the survival of hatched larvae in gynogenetic and haploid groups compared to the control group. UVirradiation of 60 s and 70 s was found to be optimal for producing viable putative gynogenetic diploids. A duration of 2 min was optimal when eggs were heatshocked at 34 °C starting in the 18th minute after fertilization to retain the second polar body and to produce gynogenetic diploids. Most of the embryos in the haploid control groups died at an early stage of development before neurulation, or they displayed features of “haploid syndrome” (abnormal body shape, open blastopore) and failed to hatch normally. Unlike haploid larvae, the obtained gynogens have started to feed, but after a few days the water condition drastically decreased and this fact was the cause of gynogens lost. The results of microsatellite DNA analysis showed that there was no genetic contribution from the paternal
Fig. 1. Survival of gynogenetic, haploid and control groups of hybrid sterlet × bester. A — haplo 1, haplo 2, haplo 3 — sperm inactivation through 45 s, 60 s and 70 s respectively; B — gyno 1, gyno 2, gyno 3 — sperm inactivation through 45 s, 60 s and 70 s respectively; x-axis: days post-hatch.
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57
Fig. 2. Afu-68 allele segregation in gynogenetic offspring of sterlet (Acipenser ruthenus). M1 — marker 25 bp (Promega), M2 — marker ϕX 174 (Promega), gynogenetic offspring — samples: 1–13, female of sterlet — sample 14, male of beluga × sterlet — sample 15.
genome in the gynogenetic group. Locus Afu-68 was monomorphic in studied groups of gynogenetic and haploid fish, and an allele 204 base pairs (bp) was observed in those groups of fish (Fig. 2). The allele 204 bp was characteristic for the mother of the gynogenetic offspring, while the male (sperm donor) exhibited alleles of 230 and 136 bp. Loci Afu-19 and
Aox-45 were polymorphic in studied gynogenetic groups of sterlet, because of the heterozygous state of the gynogenotes' mother. At locus Afu-19, two alleles of 138 and 141 bp were observed in diploid gynogens of sterlet, while at locus Aox-45, alleles of 133 and 145 bp were observed. The sperm donor possessed two alleles of locus Afu-19 (165 and 138 bp) and one allele of locus Aox-45 (121 bp). The cytogenetic analysis showed that the chromosome number in all studied gynogenetic individuals was diploid (∼ 120 chromosomes in metaphase plates) (Fig. 3). 4. Discussion
Fig. 3. Metaphase chromosome set from diploid gynogenetic sterlet (Acipenser ruthenus).
Induction of gynogenesis and polyploidy in sturgeon species is of interest to commercial aquaculturists and to researchers investigating genetics and the mechanism of sex determination in fishes. In sturgeons, the induction of gynogenesis would be important for potential production of all-female populations for caviar production. In the present research, the production of gynogenetic sterlet was described. The haploid embryos displayed the same “haploid syndrome” as was
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described by Van Eenennaam et al. (1996) in white sturgeon. Irradiation shock duration of 60 s and 70 s was optimal for bester sperm that was used for fertilization of eggs of sterlet. The UV-irradiation to deactivate the male genome prior to gynogenesis created some difficulties due to the small size of bester spermatozoa with acrosomes. The spermatozoa of the sterlet (and probably bester, too) are more sensitive to UV exposure than spermatozoa of the other sturgeons (Recoubratsky et al., 2003). However, the failure of sterlet gynogenesis in the study of Recoubratsky et al. (2003) was probably caused by too strict conditions for heat shock of the eggs (37 °C for 2.5 min in comparison to 34 °C for 2 min in the present study). A method to provide genetic verification of gynogenesis in progeny was shown. Microsatellite DNA analysis revealed that individuals from haploid (haplo 2 and haplo 3) and gynogenetic (gyno 2 and gyno 3) groups had no apparent paternal inheritance. The results of this study may be useful for future research. Irradiated bester (and probably sterlet) sperm could be useful for gynogenesis induction in other sturgeon species (as a heterologous sperm). Acknowledgments The study was supported by the University of Olsztyn grant no. 080302.0205. We thank Ms. Elzbieta Fopp and Mr. Andrzej Fopp from the Wasosze fish farm for kindly providing fish for the study. We also thank the anonymous reviewers for useful comments on the manuscript. References Arai, K., 2001. Genetic improvement of aquaculture finfish species by chromosome manipulation techniques in Japan. Aquaculture 197, 205–228. Chebanov, M., Billard, R., 2001. The culture of sturgeons in Russia: production of juveniles for stocking and meat for human consumption. Aquat. Living Resour. 14, 375–381. Dettlaff, T.A., 1993. Sturgeon fishes. In: Dettlaff, T.A., Ginzburg, A.S., Schmalhausen, O.L. (Eds.), Developmental Biology and Aquaculture. Springer-Verlag, Berlin, pp. 67–71. Grande, L., Bemis, W.E., 1991. Osteology and phylogenetic relationships of fossil and recent paddlefish (Polyodontidae) with comments on interrelationships of Acipenseriformes. J. Vertebr. Paleontol. 11 (suppl. No 1) 121 pp.
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