National Research Institute of Aquaculture, Nansei, Mie 516-08, Japan
ABSTRACT Sexual behavior of triploid males of masu salmon (the Amago strain, Oncorhynchus masou rhodurus) was observed to determine whether they showed quivering, a typical courtship behavior of salmonids, and if this behavior could induce spawning behavior and oviposition of diploid females. The triploid males showed external secondary sexual characteristics without sperm, while the diploids were spermiating. A triploid or diploid male was placed in an aquarium with an ovulated diploid female, and the behavior of male fish was observed. Triploid males showed typical quivering toward ovulated diploid females as did diploid males. In the first experiment, the mean frequencies of quivering in the triploids and diploids were 83.6/h and 40.7/h, respectively. However, females failed to spawn with both diploid and triploid males. In the second experiment with slight modification of the aquarium conditions, the frequencies of quivering in triploid and diploid males were 89.0/h and 75.8/h, respectively. All the females tested spawned accompanied by spawning behavior of males, irrespective of the male ploidy. These results indicate that triploid male masu salmon without sperm show typical courtship behavior and have the ability to induce spawning of ovulated females, suggesting that a female could spawn with a triploid male even in a natural environment.
INTRODUCTION Technical development of chromosome set manipulation has promoted not only experimental but also practical uses of triploid fish whose performances are expected to be better than those of diploids, due to their lower investment in gonad genesis (Lincoln and Bye 1984). In general, although triploid females have poorly developed ovaries, triploid males in some salmonids have been known to have partially developed testes with normal levels of reproductive hormones (Benfey and Sutterlin 1984, Lincoln and Scott 1984, Johnson et al. 1986, Kobayashi 1992, Kobayashi et al. 1993). Also, triploid females of masu salmon are sterile, and the testes of triploid males are less developed than diploid males and have no spermatozoa even in the breeding season (Nakamura et al. 1987). However, the males show external secondary sexual characteristics with similar levels of steroid hormones and gonadotrophic hormone (GTH) as diploid males. These facts lead us to assume that triploid males, even without sperm, show typical sexual behavior toward diploid females. The sexual behavioral pattern of triploid fish has not been fully investigated. Prior to actual use of triploid fish for fish farming, their reproductive potentialities should be fully investigated, and the risk of their escape into a natural environment should be assessed. In this study, sexual behavior of triploid males of masu salmon was observed to determine whether males show quivering, a typical courtship behavior of salmonids, and if this behavior would induce spawning behavior and oviposition of diploid females. Part of this paper has been presented earlier in a preliminary form (Kitamura et al. 1991).MATERIALS AND METHODS
The test animals used in this study were masu salmon of the Amago strain, Oncorhynchus masou rhodurus. Fish were raised on a commercial trout diet in an indoor glass tank (30 x 60 x 30 cm in size; water temp., 13-16oC), and in an outdoor pond (1 x 4 x 1 m in size; water temp., 13-16oC). Triploidy was induced by hydrostatic pressure at 650 atm for a duration of 6 minutes, 15-30 minutes after fertilization (Onozato 1983). Triploids were identified just prior to the experiments by comparing major axes of red blood cells (RBC) with those of diploid controls, using an image analyzer (Ibas 2000, Zeiss Co. Ltd.). The following two experiments were conducted in 1989 and 1990.
In the first experiment, triploid males were 2 years old and diploids were yearlings. The mean values of RBC major axes of triploids and diploids were 18.5-20.0 mm and 14.8-15.8 mm, respectively. The body lengths of triploids and diploids ranged from 25.0 to 28.5 cm and from 19.0 to 24.5 cm, respectively. The test tank was 45 x 90 x 45 cm in size, with a sufficient depth of gravel to allow for nest building. Precooled well water (15oC) was provided at a velocity of about 3cm/s through the tank during the experiments. A triploid or diploid male was placed in the tank with a diploid-ovulated female (2 years old, body length 22.8-28.8 cm), and then male behavior was observed and recorded using a video camera recorder for a constant time. Frequencies of quivering displayed by males were counted for 1 hour from the time of the first quiver, and subsequently, whether or not spawning occurred, was observed for 24 hours. Ten different pairs of diploid male-diploid female and five pairs of triploid male-diploid female were used. In the first experiment, we failed to induce female spawning, and then conducted the following experiment. Both triploid and diploid males were yearlings. Body lengths of the triploids and the diploids were 12.5-18.9 cm and 17.0-18.3 cm, and the average major RBC axes were 18.3 mm and 16.9 mm, respectively. Diploid-ovulated females paired with the males were 2 years old and 23.0-28.5 cm in body length.
In the second experiment, we modified aquarium conditions by changing tank size and water velocity to promote induction of oviposition. The tank size was changed to 45 x 120 x 45 cm, and the velocity was increased to 12 cm/s. Five pairs of diploid male-diploid female and three pairs of triploid male-diploid female were used. Other procedures were the same as those above. Differences in the frequencies of quivering between the triploids and the diploids was statistically evaluated by the Mann-Whitney U-test (p<0.05).
RESULTS AND DISCUSSION
Although the triploid males used in this study showed external secondary sexual characteristics, their testes appeared to have almost no sperm. By contrast, the diploids were spermiated and sperm was easily expelled by slight pressure to the abdomen. Based on histological examinations (photographs not shown), almost no sperm was found in the seminal lobules of the triploid testes which is known to be involved in the production of steroid hormones, while seminal lobules in the diploid testes were filled with sperm. Although we did not determine steroid hormone levels in the present study, Nakamura et al. (1987) reported that steroid homone and GTH levels in triploid male masu salmon are not significantly different from those in diploid male, as is the case of other salmonids (Benfey and Sutterlin 1984, Lincoln and Scott 1984, Johnson et al. 1986, Kobayashi et al. 1993).
Triploid males showed typical quivering behavior toward ovulated-diploid females as did diploid males. As reported previously (Kitamura et al. 1991), male masu salmon showed two different patterns of quivering (Fig. 1). The total numbers of the two types of quivering are shown in Table 1. The triploid and diploid males both began their quiverings 10 to 30 minutes after being placed in the tank. In the first experiment, the mean frequencies of quivering in the triploids and the diploids were 83.6/hr (range, 54-122/hr) and 40.7/hr (range, 9-79/hr), respectively. The former number was significantly higher than the latter. A possible reason that triploid males quivered more than twice as much as diploids might be attributable to differences in the age between the triploids and diploids rather than in ploidy differences. In the first experiment, female spawning failed to be induced, although nest digging behavior by ovulated females was often observed. In the second experiment where the aquarium conditions were slightly modified, the triploids displayed a frequency of quivering similar to the diploids (Table 1). The frequencies of quivering in triploid and diploid males were 89.0/hr (range, 39-141/hr) and 75.8/hr (range, 34-109/hr), respectively, without significant differences. All the females tested spawned within 12 hours after the beginning of the experiments, irrespective of the male ploidy. However, at the moment of spawning, the triploids showed spawning acts without sperm release, while sperm release was observed in the diploids. Normal display of sexual behavior by triploid male masu salmon in these experiments can probably be ascribed to the natural activity of steroid hormone productions within the fish (Nakamura et al. 1987).
Thus, in this study, we confirmed that triploid male masu salmon without sperm show typical courtship behavior and have the ability to induce spawning of ovulated females. Similarly, Inada and Taniguchi (1991) observed that induced triploid males of ayu (Plecoglossus altivelis) with poorly developed testes chased female diploids and were involved in spawning activity. These investigations suggest that a diploid female could spawn with triploid males even in a natural environment. However, mating by normal females and triploid males, even if they have sperm that have the ability to fertilize, would result in abnormal development of the zygote. Escape or release of a large number of induced triploid males into a natural environment could affect the productivity of the natural resources.
ACKNOWLEDGMENTS
We are grateful to Dr. Jeffrey Silverstein of National Research Institute of Aquaculture, Japan, for his critical reading of the manuscript.
LITERATURE CITED
Benfey, T.J. and A.M. Sutterlin, 1984. Growth and gonadal development in triploid landlocked Atlantic salmon (Salmo salar). Can. J. Fish. Aquat. Sci. 41:1387-1392.
Inada, Y. and N. aniguchi, 1991. Spawning behavior and after-spawning survival in induced triploid ayu (in Japanese) Plecoglossus altivelis. Nippon Suisan Gakkaishi 57: 2265-2269.
Johnson, O.W., W.W. Dickhoff, and F.M. Utter, 1986. Comparative growth and development of diploid and triploid coho salmon, Oncorhynchus kisutch. Aquaculture 57:329-336.
Kitamura, S., H. Ogata, and H. Onozato, 1991. Triploid male masu salmon Oncorhynchus masou shows normal courtship behavior. Nippon Suisan Gakkaishi 57:2157.
Kobayashi, T., 1992. Growth, survival and reproductive cycle of induced triploid rainbow trout under the communal rearing condition with diploid for long period (in Japanese). Suisanzoshoku 40:57-70.
Kobayashi, T., N. Sakai, S. Fushiki, Y. Nagahama, M. Amano, and K. Aida, 1993. Testicular development and changes in the levels of reproductive hormones in triploid male rainbow trout (in Japanese). Nippon Suisan Gakkaishi 59:981-989.
Lincoln, R.F. and V.J. Bye, 1984. Triploid rainbow trout show commercial potential. Fish Farmer 7:30-32.
Lincoln, R.F. and A.P.Scott, 1984. Sexual maturation in triploid rainbow trout, Salmo gairdneri, R. J. Fish. Biol. 25:385-392.
Nakamura, M., F. Tsuchiya, M. Iwabashi, K. Suzuki, and Y. Nagahama, 1987. Oral presentation at Jpn. Soc. Sci. Fish. Biennial Meeting in Spring 1987 (Abstr. 323, in Japanese).
Onozato, H., 1983. Diploidization of gynogenetically activated salmonid eggs using hydrostatic pressure. Aquaculture 43:91-97.
¹Present address:Nikko Branch, National Research Insititute of Aquaculture, Nikko, Tochigi 321-16, Japan.
²To whom all correspondence should be addressed.
