Induced interspecific androgenesis using diploid sperm from allotetraploid hybrids of common carp×red crucian carp

Aquaculture 264 (2007) 47 – 53 www.elsevier.com/locate/aqua-online

Induced interspecific androgenesis using diploid sperm from allotetraploid hybrids of common carp×red crucian carp Yuandong Sun 1 , Chun Zhang 1 , Shaojun Liu ⁎, Wei Duan, Yun Liu College of Life Sciences, Hunan Normal University, Changsha 410081 Hunan, People's Republic of China Received 23 January 2006; received in revised form 7 July 2006; accepted 7 July 2006

Abstract In the present study, viable diploid androgenetic individuals (AT-ag) (2n = 100) have been developed in UV-radiated eggs of goldfish (GF) (Carassius auratus) following fertilization with diploid spermatozoa from male allotetraploid hybrid (AT) F13 (4n = 200) of red crucian carp (C. auratus red var.) (♀) × common carp (Cyprinus carpio L.) (♂). In the experimental groups, eggs were inactivated by UV-rays, the range of UV-radiation dose tested varied from 240 to 600 mJ/cm2, the relative high hatching rate (4.1 ± 0.4%) and survival from hatching to start of feeding (41.3 ± 2.6%) were given at UV dose of 300 mJ/cm2. The androgenetic nature was confirmed by chromosome observation and DNA content measurement of somatic cells by a flow cytometer. The AT-ag were diploid (2n = 100) whereas the controls (GF × AT) were triploids (3n = 150). The diploid AT-ag grew slower than the triploid GF × AT. The AT-ag and GF × AT were also morphologically distinguishable. The AT-ag began to sexually mature at the age of 2 years, and their fertility further increased at the age of 3 years. The tetraploids were obtained in the offspring (F1ag × ag) produced by mating the males with females of AT-ag and in the progeny (AT-ag × AT) generated by crossing the males of AT with the females of AT-ag, indicating that diploid AT-ag could produce diploid gametes. All the sex ratios of males to females in AT-ag, F1ag × ag, ATag × AT were not significant (P N 0.05) from the ratio of 1:1, suggesting the XXXY sex-determining mechanism in the males of AT. The present study indicated that bisexual fertile diploid AT-ag were successfully induced from diploid spermatozoa of AT. © 2006 Elsevier B.V. All rights reserved. Keywords: Androgenesis; Allotetraploid hybrid; Diploid sperm; Haploid egg; Goldfish

1. Introduction Androgenesis is an induced developmental process enabling for the exclusive paternal inheritance of nuclear genome. It involves inactivation of the female genome using irradiation (gamma, X or UV-rays) followed by fertilization with spermatozoa. To obtain ⁎ Corresponding author. Tel.: +86 731 8873010; fax: +86 731 8873074. E-mail address: [email protected] (S. Liu). 1 These authors have equally contributed to this work. 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.07.004

viable androgenetic individuals, the paternal haploid chromosome set must be doubled using physical shocks. However, when diploid spermatozoa are used, androgenetic diploids can be produced without any treatment for chromosome duplication (Thorgaard et al., 1990; Arai et al., 1995). Important factors in successfully induced androgenesis include optimum irradiation dose applied to the eggs, the quality of the eggs, and the type, duration and time of initiation of physical shocks. Androgenesis has been successfully induced in a number of species, such as Cyprinus carpio (Bongers et al., 1995; Grunina et al., 1995), Oncorhynchus mykiss

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(Thorgaard et al., 1990; Scheerer et al., 1991), Misgurnus anguillicaudatus (Arai et al., 1995), and Misgurnus mizolepis (Nam et al., 2000). Most studies on androgenesis used diploid fish (Babiak et al., 2002a,b; Stoss, 1983; Parsons and Thorgaard, 1985). Reports on androgenesis using diploid sperm are rare (Thorgaard et al., 1990; Arai et al., 1995; Morishima et al., 2004). Survival of androgenetic individuals is generally low due to the drastic treatments applied to eggs before insemination and to the zygotes at the first mitotic division (Chourrout, 1986; Pandian and Koteeswaran, 1998; Yamazaki et al., 1993). The insemination of irradiated eggs with diploid spermatozoa of artificially induced tetraploid fish is an alternative method for improving the survival of androgenetic individuals. In our previous studies (Liu et al., 2001, 2004a; Sun et al., 2003), F1–F2 hybrids of the red crucian carp (C. auratus red var.) (♀) × common carp (C. carpio L.) (♂) were proved to be diploid (2n = 100). These F2 hybrids produced diploid sperm and diploid eggs to form allotetraploid F3 hybrids. Since then, consecutive allotetraploid generations have been produced, and recently the F16 allotetraploid generation was established. In F3–F16 allotetraploid generations, both female and male allotetraploid hybrids were fertile. During recent years, more than 100,000 allotetraploid hybrids were annually produced, providing an abundant source of diploid spermatozoa. In the present study, we attempted to produce androgenetic individuals by inseminating UV-irradiated eggs of goldfish with spermatozoa from male allotetraploid hybrids of red crucian carp (♀) × common carp (♂). We identified the ploidy status of the androgenetic individuals by chromosome counting and DNA content measurement. The aim of our study was to investigate the survival and growth of androgenetic allotetraploid hybrids and to investigate the mechanism of ploidy formation in androgenetic progenies of allotetraploid common carp–crucian carp hybrids. 2. Materials and methods 2.1. Eggs and spermatozoa Four one-year-old female goldfish (GF) (A, B, C and D) were obtained from a commercial dealer and used on the same day. The haploid eggs were hand-stripped from the females 10–12 h after injection with human chorionic gonadotropin (HCG, 1–1.5 μg/g body weight). Eggs of female-B were overripe. Diploid spermatozoa were obtained from two 1-yearold F13 allotetraploid male hybrids (AT) of red crucian carp (♀) × common carp (♂), which came from the

National Tetraploid Population Protection Station located in Hunan Normal University, China. The milt of the two AT males was mixed and diluted with Hank's solution (1:4), then stored at 4 °C for use. 2.2. Induction of androgenetic development Eggs of female GF were irradiated with UV-rays to inactivate the maternal genome. The egg samples were kept in plastic containers on ice, and about 5 ml of synthetic ovarian fluid was added to each container to obtain a single layer of eggs. Synthetic ovarian fluid consisted of 4.11 g BSA per liter, 3.8 mmol Na2HPO4, 118.0 mmol NaCl, 12.7 mmol KCl, 0.7 mmol MgCl2·6H2O, 2.7 mmol CaCl2, 5.5 mmol tyrosine and 5.5 mmol glycine in distilled water (Bongers et al., 1994). The pH was adjusted to 8.14 with NaHCO3. The eggs from each fish were divided into six batches and each batch was further divided over three plastic containers (200–400 eggs per container). Five batches were irradiated by UV-rays and one batch was used as the control without UV-irradiation. During irradiation, the eggs were stirred by a rocking machine to ensure a homogenous irradiation of all eggs. The range of total UV dosages tested varied from 240 to 600 mJ/cm2, which corresponded to irradiation durations of 120 s to 300 s. After irradiation, eggs were immediately fertilized with AT milt to form the androgenetic individuals (AT-ag). The untreated eggs were inseminated with the same milt to form the control group (GF × AT). None of the zygotes were subjected to physical shocks after insemination. 2.3. Incubation and rearing Fertilized eggs were incubated at 18–20 °C in fresh water until hatching. Hatched fry were kept in 58 plastic basins according to the different female origin and UV dose. Hatching rates and survival from hatching to first feeding were recorded for each batch. At first feeding all fry from the same UV or control treatment (240, 300, 360, 420 and 480 mJ/cm2 respectively) were pooled and reared in a single pond. In total six ponds were used for ongrowing. The numbers and morphology of the fry of each group were examined one month after stocking. 2.4. Sexuality and fertility During each reproductive season, all AT-ag and GF × AT fish were examined to verify whether they produced eggs or semen. Sexually mature AT-ag animals were first found at age of 2 years. In 2005, offspring of ATag fish (F1ag × ag) were produced by crossing AT-ag males with AT-ag females. AT-ag × AT progeny were generated

Y. Sun et al. / Aquaculture 264 (2007) 47–53

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Table 1 Hatching rates in relation to the females and the UV dose administered UV dose (mJ/cm2)

Females

240a A⁎ B⁎⁎ C⁎ D⁎

3.7; 3.9; 1.7; 1.9; 3.4; 3.6; 3.4; 3.5;

4.1 2.1 3.9 3.8

300a

360a

3.8; 4.0; 4.5 1.2; 1.4; 1.5 3.6; 3.7; 4.0 3.4; 3.6; 3.9

3.1; 3.4; 1.1; 1.2; 2.9; 3.1; 2.7; 2.9;

3.7 1.4 3.3 3.0

420ab

480b

2.4; 2.8; 2.9 0.6; 0.7; 0.9 2.1; 2.3; 2.4 1.9; 2.1; 2.2

0.6; 0.0; 0.4; 0.5;

0.9; 1.0 0.0; 0.4 0.5; 0.6 0.7; 0.7

⁎Indicating under the same UV dose, hatching rates of different female do not differ significantly (P N 0.05). ⁎⁎Indicating the hatching rates of female B were significantly lower than the rates of other females under the same UV dose (P b 0.05). The same superscript indicated that the hatching rates of other female (A, B, and C) do not differ significantly (P N 0.05). Hatching rate = (number of hatching fry / number of eggs) × 100%.

by crossing AT males with AT-ag females. In 2006, F1ag × ag were again produced by mating 3-year-old AT-ag males and females. 2.5. Chromosome observation and DNA content measurement To determine the ploidy level, 1 Goldfish, 5 putative AT-ag fish (1 fish per pond), 5 large individuals (1 fish per pond), 20 F1ag × ag fish, 7 AT-ag × AT fish and 10 control GF × AT fish were sampled. The chromosome preparations of kidney cells in the above fish and chromosome spreads of spermatogonia in 1-year-old AT-ag were obtained according to the procedure described by Zhang et al. (2005). Chromosome spreads were observed by a light microscope and photographed with Pixera Pro 600ES (Pixera Corporation, U.S.A.). In each sample, at least 50 metaphase spreads per fish were counted. One red crucian carp (C. auratus: RCC), and the above mentioned fish examined for chromosome observation were sampled for the DNA content measurement. Tissues of caudal fin were collected, minced, and treated with nuclei extraction (Partec, Germany). Samples were then filtered, and stained with DAPI DNA staining solution

(Partec, Germany). The DNA content of each sample was measured using a flow cytometer (Cell Counter Analysiser, Partec, Germany). The DNA content of the diploid red crucian carp (2n = 100) was used as the standard. 2.6. Statistical analysis Relative hatching rate and survival from hatching to start of feeding of each experimental group were statistically analyzed using one-way ANOVA (LSD and Duncan) (P b 0.05). Hatching rates were expressed as (number of fry at hatching / total number of eggs incubated) × 100%, survivals from hatching to feeding were calculated as (total number of normal fry at start of feeding / total number of fry at hatching) × 100%. All analyses were carried out using SPSS 11.0 software (SPSS Inc. U.S.A.). The sex ratios of AT-ag progeny groups, 1-year-old F1ag × ag progeny (2005 and 2006), and AT-ag × AT Table 3 Survival after one month and the ratio of small individual in different ponds Pond

Table 2 Survival from hatching to start of feeding in relation to the female and the UV dose administered Female

A B C D Pond

Survival Large after one individuals month (%) after five months

N

Mean weight (g)

75.7

4 105 ± 14

7

55 ± 8

75.0

4

92 ± 11

14 60 ± 12

77.8

69.5

3

89 ± 9

9

48 ± 10

75.0

62.4

2

85 ± 11

5

50 ± 9

71.4

67.0

3 111 ± 13

2

76 ± 5

40.0

UV dose (mJ/cm ) 300a

360a

420b

480b

39.3 ± 1.1 32.1 ± 0.9 33.5 ± 2.4 40.7 ± 3.0 1#

41.3 ± 2.6 27.4 ± 1.3 38.6 ± 0.7 39.1 ± 0.9 2#

36.9 ± 1.0 12.8 ± 1.2 37.4 ± 0.9 32.7 ± 2.3 3#

23.0 ± 3.5 19.0 ± 2.0 20.1 ± 1.4 19.5 ± 0.8 4#

16.5 ± 4.7 0 24.6 ± 5.1 23.4 ± 3.3 5#

The same superscript indicated that the survivals from hatching to start of feeding of different female were not significant difference (P N 0.05). Survivals from hatching to start of feeding expressed as (number of normal fry at start feeding/ total number of fry at hatching± SD) × 100%.

1# (240 mJ/cm) 2# (300 mJ/cm) 3# (360 mJ/cm) 4# (420 mJ/cm) 5# (480 mJ/cm)

Rate of small individual (%)

N Mean weight (g)

2

240a

Small individuals after five months

63.6

Survival after one month was calculated as (number of individual after one month / number of normal fry at start feeding) × 100%.

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Table 4 Comparison of morphology characteristics in GF, AT-ag, AT and GF × AT GF Body length/ body height Dorsal fin formula Barbel Lateral scale formula

AT

AT-ag

GF × AT

0.73 ± 0.05 0.39 ± 0.10 0.41 ± 0.06 0.50 ± 0.13 I + 17 III + 19 III + 18 II + 18 No barbel Two pairs Two pairs One pair 25–26 6/7 30–34 6/7 31–33 6/7 30–31 6/7

progeny were examined, respectively. All sex ratios were tested for deviation from a 1:1 ratio using an X2 test. 3. Results

After one month, the survival rates of the experimental groups averaged 75.7%, 75.0%, 69.5%, 62.4% and 67.0% (expressed as the number of fry after one month / number of fry at first feeding) for UV dosage 240, 300, 360, 420 and 480 mJ/cm2, respectively; survival in the control group was 79.3%. In appearance, all GF × AT had similar morphology. In the experimental groups, two morphology types of individuals were observed. “Large” individuals had a higher mean weight and were morphologically similar to GF × AT fish, whereas “small” individuals had a lower mean weight and were morphologically similar to allotetraploid AT-F13 fish (Tables 3 and 4). In experimental groups, the ratios of the number between large individuals and small ones varied from 40 to 77.8%.

3.1. Survival and morphology Hatching rates of GF × AT were 87.6%, 47%, 87.2% and 86.9% in female A, B, C, and D, respectively. The low hatching rate of eggs from female B suggests poor egg quality. In experimental groups, with different irradiation duration, the hatching rates and survival from hatching to start of feeding varied (Tables 1 and 2, respectively). Under the same UV-irradiation, the hatching rates of eggs from female A, C and D were not significantly different (P N 0.05), and significantly higher than of eggs from female B (P b 0.05). The hatching rates of eggs treated at UV dose of 240, 300 and 360 mJ/cm2 didn't differ significantly (P N 0.05), but were significantly higher than the hatching rates of eggs treated with a UV dose of 420 mJ/cm2 or 480 mJ/cm2 (P b 0.05; Table 1). The survival rates from hatching to first feeding of groups treated with UV dose 240, 300 and 360 mJ/cm2 were not significantly different (P N 0.05) from each other, and significantly higher than of the groups receiving UV dose 420 and 480 mJ/cm2 (P b 0.05; Table 2). Less than 0.1% viable individuals were obtained when irradiation dose was 600 mJ/cm2 (data not shown in Table 2). The highest hatching rate (mean 4.1%) and survival from hatching to start of feeding (41.3 ± 2.6%) were recorded for female A at a UV dose of 300 mJ/cm2.

3.2. Chromosome observation and DNA content measurement The chromosome number of GF fish ranged from 95 to 100; 80.0% of the chromosome spreads had a diploid number of chromosomes (2n = 100) (Table 5). The chromosome number of “small” individuals (AT-ag) ranged from 95 to 100 with 91.2% of metaphase chromosome spreads containing 100 chromosomes (Table 5). “Large” individuals were triploid with 150 chromosomes (3n = 150), suggesting unsuccessful activation of maternal genome. In the control group, all examined individuals (GF × AT) were triploid (3n = 150), their chromosome number ranged from 145 to 150 (90.6% 150; Table 5). The F1ag × ag and AT-ag × AT groups were tetraploid (median 4n = 200), with chromosome numbers ranging from 195 to more than 200 (Table 5). In the experimental groups, only the small individuals with 100 chromosomes were regarded as putative AT-ag (Fig. 1). Metaphase chromosome spreads of spermatogonia in 1-year-old AT-ag fish contained 100, 200 or more than 200 chromosomes. The percentage of spreads with 200 chromosomes accounted for 30.7%. The results of the distribution of DNA content of the samples are shown in Table 6. DNA of diploid RCC

Table 5 Distribution of chromosome numbers for GF, AT-ag, GF × AT, F1ag × ag and AT-ag × AT Fish type

GF AT-ag GF × AT F1ag × ag AT-ag × AT

Sample numbers

Spread numbers

Chromosome number b95

95–100

b145

145–150

1 5 10 20 7

50 250 500 1000 350

10 22

40 228

0 0 47

453

b195

195–200

N200

97 53

878 285

25 12

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(2n = 100) was used as the standard. The results show that the DNA content of AT-ag fish was slightly larger than that of diploid GF, and two-thirds that of GF × AT fish.

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Table 6 The distribution of DNA contents of RCC, AT-ag, GF and GF × AT Fish type

Mean DNA content

RCC AT-ag GF × AT GF

73.04 84.90 103.19 77.99

Ratio Observed

Expected

(AT-ag/RCC) 1.16 ((GF × AT)/RCC) 1.41 (GF/RCC) 1.07

1 1.5 1

The observed ratio was not significantly different (P N 0.05) from the expected ratio.

The DNA content of “large” individual in experimental groups was equal to that of GF × AT. These results were in accordance to chromosome number evaluation. 3.3. Fertility and sexuality AT-ag Females produced normal eggs with the mean diameter of 0.17 cm, which was similar to the diploid eggs of gynogenetic progeny of AT. The 2-year-old male AT-ag fish produced water-like semen, just like that of F2 hybrids of red crucian carp × common carp described by Liu et al. (2001). However, the males of the 3-yearold AT-ag fish produced white semen, suggesting that with increasing age, the fertility of the AT-ag males was improving. In 2005, about 500 live progeny were obtained by crossing 2-year-old AT-ag males and females; in 2006, more than 3000 live progeny were obtained by crossing 3-year-old AT-ag males and females. The sex ratios of males to females in AT-ag, F1ag × ag and ATag × AT progenies were 1.14, 0.82 and 1.22, respectively, which were not significantly different from a 1:1 sex ratio. 4. Discussion

Fig. 1. The metaphase chromosome spreads of an AT-ag, GF × AT and F1ag × ag. (A) The metaphase chromosome spread of an AT-ag (2n = 100). Bar = 3 μm. (B) The metaphase chromosome spread of GF × AT (3n = 150). Bar = 3 μm. (C) The metaphase chromosome spread of F1ag × ag (4n = 200). Bar = 3 μm.

Bongers et al. (1994) demonstrated that the success in inducing androgenesis contributed to the genetic nature of the sperm, egg quality and optimum UVradiation procedure. In the present study, the poor egg quality (such as female B) and the higher UV dose resulted in lower survival. The relative high hatching rate, survival from hatching to start of feeding, survival after 1 month and rate of small individual (AT-ag) were given at UV dose 300 mJ/cm2, indicating that it was the relative effective UV dose. The presence of large individual (hybrid fish) was due to the failure of inactivation of female genome, because the stickiness of the goldfish's eggs prevented the animal poles of all the eggs from being well irradiated, and also resulted in the chromosome abnormalities which were responsible for low survival (Yamazaki et al., 1993). In the present

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study, diploid AT-ag were successfully induced from diploid spermatozoa originating from AT and the androgenetic nature was confirmed by morphological characteristic and chromosome counting. In the present study, the interspecific AT-ag was induced between GF and AT, which possessed different numbers of chromosome. So the chromosome number of individual was the irrefutable proof to distinguish ATag from GF × AT. AT-ag possessed normal gonad, while all GF × AT possessed abnormal gonads (Liu et al., 2004b). With this difference, the nature of AT-ag could also be proved. On the other hand, the morphological differences between AT-ag and GF × AT were also the markers to conform the genetic origin of AT-ag. The shape of AT, which possessed two sets of chromosomes from red crucian carp and two sets of chromosomes from common carp (Liu et al., 2001), was intermediate to their original parents (red crucian carp and common carp). The shape of AT-ag was similar to that of AT and different from red crucian carp or common carp, indicating that the genetic composition of AT-ag was heterozygous consisting of one set of chromosomes from red crucian carp and one set from common carp. Both male and female AT-ag were fertile, and they mated each other to produce tetraploid F1ag × ag; on the other hand, tetraploid AT-ag × AT offspring were also obtained, which suggested that the diploid AT-ag could produce diploid gamete, which was similar to diploid gynogenetic fish from AT and F2 hybrids of the red crucian carp (C. auratus red var.) (♀) × common carp (C. carpio L.) (♂) as indicated by our previous reports (Liu et al., 2001, 2004a). We had found the evidence of the unreduced diploid eggs generated from the diploid gynogenetic individuals. The same situation was observed in 1year-old AT-ag. In the spermatogonia of 1-year-old AT-ag, only the chromosomes were in the metaphase of mitosis and no bivalent chromosomes were observed, suggesting that the spermatogonia in 1-year-old AT-ag did not develop into meiosis I. The chromosome spreads with 100, 200 and more than 200 chromosomes were observed in the metaphase chromosome spreads of spermatogonia, which indicated that the diploid AT-ag had the potential ability to generate diploid gamete. The presence of 200 or more than 200 chromosomes in spermatogonia probably contributed to the mechanism of pre-meiotic endoreduplication (Liu et al., 2001, 2004a; Zhang et al., 2005). The molecular mechanism of the pre-meiotic endoreduplication will be further studied in the future. The sex ratios of males to females in F1ag × ag and AT-ag × AT were 0.82 and 1.22, respectively, which did not differ significantly from the ratio of 1:1; and the sex ratio of males to females in AT-

ag was also not significantly different from the ratio of 1:1. The results suggested the sex-determination in male AT at least included XXXY genotype. In theory, the male AT had another genotype XXYY, which is worth further studying in the future. Our previous studies (Liu et al., 2004a; Yan et al., 2005) indicated that the female allotetraploid hybrid's sex-determination genotype belonged to XXXX because all the gynogenetic offsprings were females. In the present study, the bisexual fertile diploid AT-ag were successful obtained, providing an important platform to form the resources of diploid sperm and diploid eggs. Their bisexual fertility was a very important factor in forming a population. Both the diploid eggs and diploid sperm were very valuable in producing the parthenogenetic clone, and it is expected that the diploid gametes will play an important role in building the diploid gynogenetic or androgenetic line. Based on the obtained results, it is very possible to breed the genetically improved tetraploid hybrids, which will be of great biological significance in both theory and application. Acknowledgements We want to thank Professor Hanks Komen and Professor Gideon Hulata for critically reviewing and editing this MS. This research was supported by grants from the National Natural Science Foundation of China (nos. 30330480 and 30571444), from the Program for Changjiang Scholars and the Innovative Research Team in University (no. IRT0445), from the State Key Basic Research Project of China (973 project) (no. 2001CB 109006), and from the Training Project of Excellent Young Researchers of the State Education Ministry of China (no. 200248). References Arai, K., Ikeno, M., Suzuki, R., 1995. Production of androgenetic diploid loach Misgurnus anguillicaudatus using spermatozoa of natural tetraploids. Aquaculture 137, 131–138. Babiak, I., Dobosz, S., Kuzminski, H., Goryczko, K., Ciesielski, S., Brzuzan, P., Urbanyi, B., Horvath, A., Lahnsteiner, F., Piironen, J., 2002a. Failure of interspecies androgenesis in salmonids. J. Fish Biol. 61, 432–447. Babiak, I., Dobosz, S., Goryczko, K., Kuzminski, H., Brzuzan, P., Ciesielski, S., 2002b. Androgenesis in rainbow trout using cryopreserved spermatozoa: the effect of processing and biological factors. Theriogenology 57, 1229–1249. Bongers, A.B.J., Veld, E.P.C., Abo-Hashema, K., Bremmer, I.M., Eding, EH., Komen, J., Richter, C.J.J., 1994. Androgenesis in common carp (Cyprinus carpio L.) using UV irradiation in a synthetic ovarian fluid and heat shocks. Aquaculture 122, 119–132. Bongers, A.B.J., Abarca, B.J., Doulabi, Z.B., Eding, E.H., Komen, J., Richter, C.J.J., 1995. Maternal influence on development of aridi-

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