Aquaculture International (2006) 14:3542 DOI 10.

1007/s10499-005-9012-3

Springer 2005

Induced meiotic gynogenesis in tench, Tinca tinca (L.) using irradiated heterogenic sperm
JIAXI WANG1,*, HUIJI LIU1, WENQIANG MIN1, JINGO TONG2, MIN GUAN1, YUZHANG HAN1, LUOJUN GONG1, ZHEN HUANG1, JIE REN1, JINPING ZHANG3 and HONGPING ZHENG1
Hubei Province Fisheries Research Institute, 116 Donghu Road, Wuchang, 430071, Hubei, P.R. China; 2Academia Sinica, Institute of Hydrobiology, Wuhan, 430072, P.R. China; 3Zhaoqing Goldeatech Co. Ltd, Zhaoqing, 526200, P.R. China; *Author for correspondence (e-mail: jiaxi168@ public.wh.hb.cn) Received 8 November 2004; accepted in revised form 15 July 2005
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Key words: Heterogenic sperm, Meiotic gynogenesis, Tinca tinca Abstract. Eects of the starting time and duration of cold shock, as well as the source of heterogenic sperm on the percentage of viable gynogenic larvae (PVGL) in tench were studied. The DNA in sperm of red common carp (Cyprinus carpio var singuonensis) was inactivated by ultraviolet radiation prior to use to induce meiotic gynogenetic development in tench. In experiment 1, tench eggs were cold-shocked for 30 min starting at 1, 3, 5, 7, 10 and 15 min post activation. In experiment 2, cold shock began 5 min after activation and lasted for 10, 20, 30, 40, 50 min, respectively. Each experiment was run in triplicate using 3 tench females, and one group not treated with cold shock was included in each experiment to serve as a control group. In experiment 3, sperm of bigmouth buffalo (Ictiobus cyprinellus), red common carp or grass carp (Ctenopharyngodon idella) was used to induce gynogenesis in tench with a 20-min cold shock starting 5 min post activation. The results showed that PVGL from the control group was very low (0.110.47%). In experiment 1, the highest average PVGL (9.60%) was observed when cold shock treatment was applied 5 min post activation. When cold shock treatment was started 5 min post activation, duration of cold shock affected PVGL. Cold shock lasting 20 min resulted in the highest average PVGL (12. 57%) among the selected duration of cold shock studied in experiment 2. The average PVGL was 2.3, 8.6, and 9.3%, respectively, for eggs induced by sperm of bigmouth buffalo, red common carp and grass carp. Average PVGL was significantly lower for eggs induced by sperm of bigmouth buffalo, compared with that for eggs induced by sperm of the other two species. However, average PVGL were similar for eggs induced by sperm of red common carp and grass carp. In summary, the optimal conditions for gynogenesis in tench include the use of irradiated sperm of grass carp to activate the eggs and cold shock of 20 min starting 5 min post activation. Since female tench grow much faster to a larger size than male tench, gynogenesis of tench holds great potential for production enhancement.

Introduction Gynogenesis is a type of sexual reproduction requiring insemination, where the nucleus of the sperms is inactivated before the sperm penetrates into the eggs, and development of the embryo is controlled solely by maternal genetic materials. Gynogenesis was rst established and developed in the fties of twentieth century. It has been widely used in genetic and breeding studies, as

36 well as for constructing inbred strains and for controlling sh reproduction in natural habitats by introducing unisexual female ospring. Of the various methods for producing gynogenetic ospring, temperature shock is the most simple and eective. When homogenous sperm was used to induce gynogenesis, incomplete inactivation of spermatozoa made it dicult to detect and to explain the results. Thus, heterogenic sperm has been widely used to induce gynogenesis. Tench Tinca tinca (L.) is a cyprinid species of commercial importance for pond aquaculture. It is native to many European countries, West Siberia and Caucasus Mountains (Banarescu 1960/61), as well as the Elcheese river of Xingjiang province in China (Sifa 1979). There were records of tench farming in Europe since the Middle Ages. However, the process of extensive reproduction and intensive domestication of this species started only recently (Billard and Flajs hans 1995). In China, the studies on tench have been scarce. Chinese tench farming commenced in 2000. It is extended to most Chinese provinces and regions till now. From the second year the tench females grow faster than the males (Nordquist 1927; Walter, 1927; Heuschmann, 1939). Therefore, the application of unisexual females could increase production efficiency and bring significant economic benefits to tench farmers. The objective of this study was to develop the techniques for gynogenesis of tench. We used heterogenic sperm of bigmouth bualo, red common carp and grass carp to induce gynogenesis in tench in successive 2 years (2002 and 2003). Viable gynogenetic larvae were obtained. In the present paper we document the results of experiments with articial induction of meiotic gynogenesis using heterogenic sperm of several sh species.

Materials and methods Fish collection Tench (Tinca tinca) and bigmouth buffalo (Ictiobus cyprinellus) were collected from Quality Fish Species Demonstration Center, Hubei Province Fisheries Research Institute of China. Red common carp (Cyprinus carpio var singuonensis) and grass carp (Ctenopharyngodon idella) were obtained from East Lake in Wuhan with the help of a local fisherman. Female tench were induced to spawn by i. p. injection with suspension of carp pituitary particles (5 mg kg)1 body weight) in physiological saline. All the male fishes were also injected with suspension of carp pituitary at 2 mg kg)1 body weight.

Meiotic gynogenesis Semen of red common carp was diluted 1:4 with refrigerated Hanks solution in a Petri dish placed on ice and it was irradiated for 35 min with a 30 W

37 ultraviolet lamp under air atmosphere according to Wu et al. (1981). Diluted milk solution (1:10) with crushed ice was poured onto the eggs to induce genome diploidization according to Linhart et al. (1989). In the first experiment, the 30-min cold shock was initiated 1, 3, 5, 7, 10 and 15 min post activation. In the second experiment, cold shock began 5 min after activation and lasted for 10, 20, 30, 40 and 50 min, respectively. One batch of eggs without cold shock served as the control group. Each experiment was replicated 3 times using 3 tench females. Then, heterogenic sperm of bigmouth buffalo, red common carp and grass carp were irradiated by gamma radiation with 60Co at a dosage of 1000 Gy in the dark and at temperature of 02 C. Eggs activated with different heterogenic sperm were cold-shocked for 20 min in the iced milk solution starting at 5 min post fertilization. This was also repeated 3 times using eggs from 3 females. Eggs from each group were incubated in a 0.6 dm3 metal incubator (1.8 m in diameter and 24 cm in height) with flowing, clean lake water at natural temperature (2022 C). The percentage of live eggs at the morula stage and PVGL capable of swimming freely were calculated as followed: (1) the percentage of live eggs at mid blastula stage=(number of live eggs at mid blastula stage/total number of eggs)100%; (2) PVGL=(Number of viable larva/Number of living eggs at mid-blastula stage)100%.

Morphological and karyological analysis Five-month-old juveniles (812 cm) were examined for their morphological and karyological characteristics. The meristic traits of gynogenetic sh measured were compared with that of the standard diploid hatched from the eggs fertilized with tench spermatozoa. Blood was used for karyological analysis following the procedures by Wu et al. (1980). At least 5 metaphase plates from each individual fish were selected. Chromosomes were classified according to Levan et al. (1964).

Results Meiotic gynogenesis Start of cold shocks: Very low PVGL (0.110.47%) was found in eggs activated with irradiated semen from red common carp if no cold shock was applied. It was much higher for the cold-shocked eggs, but it was depending on when the cold shock treatment was applied (Figure 1). Except for eggs from female 2, the best results (PVGL 9.61.91%) were observed in the groups where the cold shock was applied at 5 min post activation. One way analysis of variance (ANOVA) revealed significant difference (p<0.05) of PVGL between 3, 5, 7, and 10 min groups and control group, respectively. In contrast, PVGL values

38 did not differ significantly (p>0.05) between 1 and 15 min groups, and the control group. Duration of shocks: When cold shock was applied 5 min after activation, the average PVGL from eggs receiving a 10, 20, 30, 40, and 50 min cold shock were 4.40, 12.57, 9.93, 6.53 and 6.40%, respectively (Figure 2). Even though some difference was found among the 3 replicates, a 20-min cold shock revealed the best result. ANOVA showed that there were significant differences (p<0.05) in PVGL between 10, 20, 30, 40, 50 min cold shock operation group and control group, only 0 min group showed no significant difference (p>0.05). Without any cold shock treatment, sperm of red common carp appeared to result in relatively high fertilization rate (up to 50%) among tench eggs. However, embryos died during the late stages of development with no sh or very few sh hatched (hatching rate <0.01%).

Eects induced by heterogenic irradiated sperm When the sperm of bigmouth bualo, red common carp and grass carp irradiated by gamma radiation was used for insemination to induce meiotic gynogenesis in tench, the average percentage of viable gynogenetic larvae was 2.3, 8.6 and 9.3%, respectively (Table 1). Statistical analysis (t-test) showed that the percentage of viable larvae from eggs inseminated by sperm of bigmouth buffalo was significantly lower than that from eggs inseminated by sperm of the other two species (p<0.05). Eggs inseminated by sperm of red common carp and grass carp resulted in similar percentage of viable gynogenetic larvae.

Figure 1. Effects of the different cold shock initiation on PVGL from eggs inseminated with irradiated sperm of red common carp. (The percentage of viable gynogenetic fries was 0.110.47% when no cold shock treatment was applied to the eggs.)

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Figure 2. Effects of cold shock duration on PVGL use irradiated red common carp sperm (PVGL was 0.170.31% when no cold shock was applied to the eggs).

Embryonic development of gynogenetic tenth Up to 4582% eggs in the experimental groups developed into cleavage stage when the eggs were incubated at 2022 C. Some embryos died during the cleavage stage, as evidenced by the anomalous cell appearance and dissolved yolk sacs. Majority of the eggs went through the blastula stage and expressed obviously haploid characters, such as the S-shaped brain, bended tail or hypoplasic tail, amplication of heart cavity, non-dierentiation of yolk sac and hypogenesis of blood circulatory system. Some of the embryos were able to
Table 1. Effects of irradiated heterogenic sperm on viable gynogenetic larvae. Trial No. Heterogenic sperm Survival rate in mid-blastula stage (%) 7.5 23.7 14.8 15.38.11 33.2 28.5 42.5 34.77.12 11.2 31.6 24.8 22.510.39 Survival rate of hatched larvae (%) 0.5 3.4 2.9 2.31.55 11.3 7.9 6.7 8.62.39 7.1 12.5 8.3 9.32.84

A1 A2 A3 AverageSD B1 B2 B3 AverageSD C1 C2 C3 AverageSD

Bigmouth buffalo Bigmouth buffalo Bigmouth buffalo Red common carp Red common carp Red common carp Grass carp Grass carp Grass carp

40 hatch but the larvae displayed various deformities, resulting in the ultimate death of the larva. Viable larva could swim freely and develop further into juveniles. The maximum PVGL from live morula stage was 17.7% in this experiment.

Morphological and karyological analyses The counts for meristic traits of gynogenetic juveniles [ D III 8, A III 68, V I 9; lin. Lat.: 92115, (2933)/(1922) ], body form and body color were similar to those of standard diploid. None of the traits was related to paternal sh. All the 198 gynogenetic sh examined were females, indicating successful production of gynogenetic osprings. Chromosomal sets of gynogenetic individuals consisted of 48 chromosomes. The karyotypes displayed characters typical of the normal diploid tench karyotype: two pairs of marker elements, i.e. the largest metacentric and the largest subtelocentric chromosome pairs.

Discussion Cold shock was eective in preventing the extrusion of the second polar body, making it possible for gynogenesis. Temperature, the time of initiation and the duration of cold shock are critical to the eectiveness of cold shock. Generally, cold treatment involves lowering the temperature of fertilized eggs to below 4 C in a relatively short time. The crushed ice was used to achieve the desired temperature (Linhart et al. 1986). In general, cold shock should start between the time after gamete activation and extrusion of 2nd polar body. Preliminary study of paraffin slices of tench embryos showed that exclusion of 2nd polar body occurred 5 min after gamete activation at 22 C. This was confirmed by our first experiment, which showed that cold shock initiated 5 min post activation resulted in the highest PVGL (17.7%). Linhart et al. (1986) suggested that the earlier the time of cold shock triggered, the shorter the duration of cold shock is needed in tench. A later cold-shock needs to last longer time (4050 min). The best duration of cold shock was suggested to be 2030 min. Duration of cold shock affects the ratio of genome diploidization. Excessively long cold shock could cause severe damage to the embryos. In our study, we found that 20-min duration gave the highest average PVGL (12.57%) when it was applied 5 min post activation, which was similar to the results (16.67%, eggs were shocked 5 min after gamete activation for 25 min) reported by Linhart et al. (1989). We did not nd any males among the gynogenic ospring of tench in our experiments, what indicated homogamety of female tench. This nding was in accordance with those of Linhart et al. (1995) in tench or of Golovinskaya et al. (1974), Stanley (1976) and Gomelsky et al. (1979) in common carp and grass carp.

41 It is suggested that the nucleus of the spermatozoan, which has penetrated into the egg, undergoes inactivation in the egg plasm, thus, the development of the embryo is controlled solely by maternal heredity. The percentage of viable gynogenetic larvae induced by homogenous sperm is apparently higher than that induced by heterogenic sperm. Incomplete inactivation of spermatozoans will make it dicult to explain the results when homogenous sperm is used to induce gynogenesis, Therefore, heterogenic sperm is often used to induce gynogenesis in most studies. In this experiment, sperm of bigmouth bualo, red common carp and grass carp was used to induce gynogenesis in tench. Grass carp is the closest relative of tench since both belong to the same family Leuciscinae, while bigmouth bualo is the least related to tench. Our results indicated that the species of sh providing the heterogenic sperms had an impact on the resulting percentage of viable gynogenetic larva. Sperm of grass carp gave the best percentage of viable gynogenetic larva, indicating the heterogenic sperm of a closely related species would help to yield more viable gynogenetic larva. This is in agreement with Shu et al. (2001), who also reported the highest percentage of viable Pengze crucian carp (Carassius auratus var pengze) larvae when the heterogenous sperm was donated by a closely related Japanese crucian carp (Carassius cuvieri). Sperm of other species such as sharpfin carp (Cyprinus acutidorsalis Wang, and silver barb (Barbus lacustris Wu) was not quite as effective in inducing gynogenesis in Pengze crucian carp. It appears that best percentage of viable gynogenetic larvae could be obtained by using the sperm of fish species that belongs to the same genus or has close relationship with species the eggs of which shall be inseminated. In summary, we were successful in inducing gynogenesis in tench. Irradiated heterogenic sperm of a close relative like grass carp could be used to activate the eggs. Cold shock initiated 5 min post activation is the most eective in preventing extrusion of the second polar body leading to gynogenesis. The optimal duration of cold shock should be around 20 min. Since female tench grows much faster to a large size than male tench, gynogenesis of tench holds great potential for production enhancement.

Acknowledgments The authors wish to thank Dr Changzheng Wang from Kentucky State University, USA for critical review of the manuscript and comments.

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