Organisme de formation enregistré sous le numéro 41570143657 auprès du Préfet de la Région Lorraine Conférence ELEVAGE DE LA LOTTE (Lota lota) : Recherche et perspectives Jeudi 1 juillet 2004 au Muséum-aquarium de Nancy 34, rue Ste Catherine 54 000 NANCY Tel : 03 83 32 99 97, Fax :03 83 32 30 16 En collaboration avec le Laboratoire de Sciences Animales de L Université Henri Poincaré Nancy I. Organisé par : 1
BURBOT (LOTA LOTA L.) REPRODUCTION IN CAPTIVITY: COMPARISON OF DIFFERENT METHODS D. Kucharczyk 1, A. Mamcarz 1, R. Kujawa 1, I. Babiak 2, J. Glogowski 3, B. Sarosiek 3, R. Kowalski 3, A. Skrzypczak 1, K. Targonska-Dietrich 1, R. Chwaluczyk 4 1- Department of Lake and River Fisheries, 2 - Department of Animal Biochemistry, Warmia and Mazury University in Olsztyn, 10-718 Olsztyn-Kortowo, Poland 3 - Institute of Animal Reproduction and food research of the Polish Academy of Sciences in Olsztyn, Poland 4 Czarci Jar Hatchery, Poland Introduction Burbot (Lota lota L.) is one of the endangered fish species in Poland and in many others European countries (Maitland and Lyle, 1991). For the other way, this species has high potential to be a coldwater cultured fish. For this reason it is necessary to develop methods of artificial spawning and rearing larvae under controlled conditions. The data on spawning of burbot in captivity are very limited (Kouril et al., 1985; Linhart, 1995). In this presentation we present the results of several works containing about burbot spawning under hatchery conditions and our study about influence of temperature on burbot embryonic development under controlled conditions. Burbot spawning using GnRHa pellets. Materials and Methods Burbot spawners (more than 100 specimens) were captured from Vistula River near Gdansk (northern Poland) in October 1997 and were transported to the hatchery. Fish were kept in 1000-l tanks with controlled temperature and photoperiod. Spawners were fed with commercial trout pellets and sporadically with frozen freshwater fish meat. After photothermal manipulations (beginning of January) fish were injected with GnRH-analogue (ovopel) at doses proposed by Horvath et al. (1997). All fish were individually marked using floy tags and weighted. Oocytes were taken from females using the method described by Kujawa and Kucharczyk (1996), were sampled in vivo and placed in Serra's solution for clarification of the cytoplasm. After 5 minutes, the position of oocytes nucleus was determined using a 4-stages scale: stage 1 - germinal vesicle in central position stage 2 - early migration of germinal vesicle (less than half of radius) stage 3 - late migration of germinal vesicle (more than half of radius) stage 4 - periphery germinal vesicle or germinal vesicle breakdown (GVBD) Ripe gamete donors were anaesthetized in a solution of 2-phenoxyethanol (0.2-0.5ml per 1l). Milt was collected with plastic syringes and kept at 4 C. Females were checked twice daily between 2 and 8 days after injection. Eggs were stripped into a plastic vessel. Eggs were fertilized using dry method. For eggs fertilization only those sperm were taken which showed the motility of more than 80% of spermatozoa. Three egg samples (100-150 eggs each) from each female were mixed with 0.05 ml of pooled milt sample. Eggs were incubated at 2 C. Some spawning characteristics, such as: percentage of spermiating males and 12
ovulating females, quantity and quality of obtained gametes, were noted. Stimulation of spermiation by GnRHa pellets. Burbot breeders were captured from Vistula River near Gdansk (northern Poland) and transported to the hatchery. Fish were kept in 1000-l tanks with controlled temperature and photoperiod. Spawners were fed with commercial trout pellets and sporadically with frozen freshwater fish meat. After photo-thermal manipulations (beginning of January) fish from treated groups were injected with GnRH-analogue (ovopel) (Table 1). One ovopel pellet (average weight - 25 mg) contains a mammalian GnRH analogue (D-Ala 6, Pro 9 Net-mGnRH at a dose 18-20 µg) and metoclopramide (dose 8-10 mg) (Horvath et al., 1997). Table 1. Description of experimental groups and used doses of hormones (quantity of applied ovopel pellets). Time between injections was 24 hrs. N = 8 in each group. Description Control Group 1 Group 2 Group 3 I injection 0.9% NaCl - 0.1 pellet - II injection 0.9% NaCl 1 pellet 1 pellet 2 pellets The influence of temperature on embryonic development of burbot. Burbot spawners (more than 100 specimens) were captured from Vistula River near Gdansk (northern Poland) and transported to the hatchery. Fish were kept in 1000-l tanks with controlled temperature and photoperiod. Spawners were fed with commercial trout pellets and sporadically with frozen freshwater fish meat. After photo-thermal manipulations (beginning of January) fish were injected with GnRH-analogue (ovopel) at doses proposed by Kucharczyk et al. (1998). Egg samples (about 500 each) obtained from several females were mixed with semen collected from several males and fertilized. Egg samples were incubated at water temperatures: 2.5, 3.7, 6.5, 8.5 C. Additionally, egg samples after 10 days period of incubation at 3.7 C were transferred to higher temperatures: 6.5, 10.5 and 13 C. All samples were made in triplicates. Every few days during incubation the observation of embryonic development was made using microscope. Results and Discussion Burbot spawning using GnRHa pellets. The survival of burbot kept for 4 months in the hatchery was very high (over 97%). Only about 50% of burbot spawners ate trout pellets, whereas more than 70% individuals fed fish meat. All fish which took food developed gonads and became mature after hormonal stimulation. Less than 30% of spawners were not mature; it might be connected with spawning behaviour of this species. Pullianen and Korhonen (1993) reported that in natural burbot populations some part (sometimes about 50%) of spawners do not mature during spawning season. In present study from all stimulated specimens (over 70) viable and good quality gametes were obtained. First donors were ready to spawn 4-6 days after last injection. Males generally produced over 10 ml of semen per 1 kg of body with percentage of motile spermatozoa (Table 2.). Similar results (also highly variable) were reported by Kouril et al. (1985) and Linhart (1995) for wild matured males. Females produced a great number of small eggs (over 800 000 per kg of female). The egg survival to the eyed-egg-stage (water 13
temperature 3-4 C) after fertilization was high: average over 70% (ranging from 30 to 95%). Table 2. The results obtained after hormonal stimulation of burbot males (n = 27). Mean males weight was 270g. Sperm characteristics Range Mean SD Quantity [ml] 0.2-10.5 3.2 2.5 Spermatozoa motility [%] 20-90 76 16 Time of motility [s] 30-66 35 19 Spermatozoa concentrations [x 10 9 ] 22.8-45.0 35.9 5.8 Obtained results: high spawners survival, high percentage of matured fish, and good quality of gametes, showed that it is possible to propagate successfully burbot in captivity, under controlled conditions. Stimulation of spermiation by GnRHa pellets. All males from treated groups were spermiated, whereas in control group only 75%. Pullianen and Korhonen (1993) reported that in natural burbot populations some part Table 3. The results obtained after hormonal stimulation of burbot males. Groups were described in Table 1. Sperm characteristics Control Group 1 Group 2 Group 3 Quantity [ml/kg] 8.2 + 4.3 11.1 + 5.3 13.9 + 4.8 12.7 + 5.0 Spermatozoa motility 65 + 9 77 + 14 79 + 12 78 + 13 [%] Time of motility [s] 38 + 12 34 + 18 43 + 20 40 + 18 (sometimes about 50%) of spawners do not mature during spawning season. The mean obtained results were compiled and presented in Table 3. In treated groups males produced more milt, which characterized with better parameters. The best results were obtained when males were treated with double injections (group 3) and with highest dose of hormones (group 4). High values of SD were results of high variation. Similar results (also highly variable) were reported by Linhart (1995) for wild matured males and by Kucharczyk et al. (1998) for artificially matured fish. Comparison of different hormonal agents Burbot is a species, which can spawn easily in the captivity. On the other hand, usually time of spawning is long, and depending of the temperature and spawners origin is lasting from few to several weeks. For this reason, different methods involved on spawning synchronisation were tested and described. In this paper we present the results from few years experiments. In this time the fish were kept in the hatchery from two weeks to four months, sometimes with different regimes of feeding. Different kinds of hormonal stimulation were tested: i.e. CPE, hcg or GnRH-a and compared with control group. The biological quality of gametes, the quantity of them, as well as the latency times were recorded. After spawning, breeders were kept in captivity for few next weeks for observation their mortality. 14
Number of ovulated females 8 6 4 2 0 1 3 5 7 9 11 13 15 17 Days sponatenous hcg GnRHa Pic. 1. Differences in spawning time of burbot females. Results and Discussion The influence of temperature on embryonic development of burbot. In present study temperatures higher than 3.7 C was lethal for burbot embryonic development (Table 4). The best results (highest hatching rate and percentage of normaldeveloped embryos) were noted in lowest tested temperatures: 2.5 C. In highest tested temperature (8.5 C) all eggs were dead during first few hours of incubation. At water temperature 6.5 C eggs started to developed, but abnormalities was noted shortly after fertilization, i.e. asynchronous cell divisions. Table 4. The results of incubation burbot eggs at different water temperatures. Water temperature Incubation period [ D] Hatching rate [%] Percentage of normal developed embryos 2.5 C 82.5 89 88.6 3.7 C 108.5 82 80.7 6.5 C - 0-8.5 C - 0 - On the other hand, eggs transferred after ten-day period incubation to higher temperatures (i.e. 6.5 C), developed quite successfully (Table 5). With the increasing temperatures, the burbot embryos survival decreased. The percentage of normally developed embryos was reciprocal correlated with incubation temperature. In highest tested temperature (13 C) only about one percent of eleutheroembryo showed any abnormalities. 15
Table 5. The results of incubation burbot eggs at different water temperatures after ten-day incubation at 3.7 C. Water temperature Incubation period [ D] Hatching rate [%] Percentage of normal developed embryos 6.5 C 110.5 74 36.1 10.5 C 125.0 34 8.2 13.0 C 134.0 16 0.9 Other embryos had different abnormalities, i.e. poorly formed retina, deformed head part of body, etc. Kucharczyk et al. (1997) reported similar developing abnormalities for bream incubated at semi-lethal temperatures. General conclusions: Burbot might spawn easily in captivity without hormonal stimulation During spontaneous reproduction spawners consuming fertilized eggs Time of reproduction without hormonal stimulation was long and asynchronous The quality of gametes was strongly fluctuated Hormonal stimulation effected with synchronous spawning The quality of gametes showed less variation than in spontaneous reproduction There were no problems with spawners survival References Horvath, L., Szabo, T., Burke, J. (1997). Hatchery testing of GnRH analogue-containing pellets on ovulation In four cyprinid species. Pol. Arch. Hydrobiol., 44: 221-226. Kokurewicz B., (1970). The effect of the temperature on embryonic development of Tinca tinca (L.) and Rutilus rutilus (L.). Zoologia Polska, 20: 317-337. Kouril, J., Linhart, O., Dubsky, K., and Kvasnicka, P. (1985) The Fertility of male and female burbot (Lota lota) following stripping of ova and semen. Prace VUHR Vodnany, 14: 75-79. (In Czech with English summary). Kucharczyk, D., Kujawa, R., Mamcarz, A., Skrzypczak, A., Babiak, I., 1998. Artificial spawning of burbot (Lota lota L.) under controlled conditions. European Aquaculture Society, Special Publication, 26: 149-150. Kucharczyk, D., Luczynski, M., Kujawa, R., Czerkies, P., 1997. Effect of temperature on embryonic and Larval development of bream (Abramis brama L.). Aquatic Sciences, 59: 214-224. Linhart, O., (1995) Spermiation. semen quality and semen quantity in some species of fish. Prace VUHR Vodnany, 31: 112-123. (In Czech with English summary). Maitland, P.S., and Lyle, A.A. (1991) Conservation of freshwater fish in the British Isles: The current status and biology of threatened species. Aquatic Conservation: Mar. Freshwat. Ecosyst., 1: 25-54. Pullianen, E., and Korhonen, K. (1993) Does the burbot, Lota lota, rest years between normal spawning seasons. J. Fish Biol., 43: 355-362. 16