뱀장어의 인공종묘생산을 위한 성성숙 유도와 치어의 영양학적 연구

Abstract

본 연구에서는 성공적인 뱀장어의 인공 종묘 생산을 위한 기초 연구로 번식 내분비학적 접근을 통해 효과적인 인공 성성숙 유도와 수정 및 부화를 위한 연구를 수행하였고, 이어 사료 영양학적 접근을 통해 치어기 뱀장어의 주요 영양소 요구량을 구명하여 향후 부화자어 및 친어의 사육 관리를 위한 기초자료를 제공하고자 하였다. 먼저 뱀장어의 인공 성성숙 유도를 위한 기초연구의 일환으로 번식 내분비학적 연구를 수행하였다. 뱀장어의 효율적인 친어관리를 위한 양식산 뱀장어의 암수 성감별에 관한 연구결과에 의하면 전장에 따라 수컷은 40~50 cm 이하, 그리고 암컷은 50 cm이상의 개체인 것으로 확인되었고, 혈중 성호르몬 농도에 있어 암컷이 수컷에 비해 testosterone과 estradiol-17β의 농도가 유의하게 높은 것으로 확인되었다. 따라서 뱀장어의 전장과 혈중 성호르몬 농도는 성감별을 위한 지표로써 효과적으로 이용될 수 있음을 제시하였다. 양식산 수컷 뱀장어의 인위적 성성숙 유도에 따른 성숙 정도를 확인하기 위해 혈중 성호르몬 변동과 정도 발달 정도를 조사한 결과 수컷 뱀장어에 HCG (human chorionic gonadotropin, 1 IU/g body weight)의 반복주사에 의해 어체중과 혈중 성호르몬(T, 11-KT, DHP)의 수준이 증대됨에 따라 정자형성 및 배정이 효과적으로 유도되었고, 이를 통해 수컷 뱀장어의 인공 성성숙 유도를 위해 HCG의 반복주사가 적합한 것으로 확인되었다. 양식산 암컷 뱀장어의 인위적 성성숙 유도에 따른 성숙 정도를 확인하기 위해 혈중 성호르몬 변동과 정도 발달 정도를 조사한 결과에 의하면 암컷 뱀장어에 연어 뇌하수체 추출물(salmon pituitary extract, SPE; 20mg/fish/week)의 반복주사에 의해 어체중과 혈중 성호르몬(T, E2)의 수준이 증대됨에 따라 난황형성이 효과적으로 유도된 반면 난소에서 DHP의 합성이 미약하여 혈중 DHP는 거의 검출되지 않았다. 따라서 최종성숙 및 배란을 위해 DHP (2 μg/g body weight)를 처리해야 하고, 아울러 성공적인 배란과 양질의 알을 획득하기 위해서는 DHP의 처리시기가 중요한 요인으로 작용하는 것으로 나타났다. 이를 통해 암컷 뱀장어의 인공 성성숙 유도에 있어 난황 형성 유도를 위해 SPE, 그리고 최종성숙 및 배란을 위해 DHP가 적합한 것으로 확인되었다. 뱀장어의 종묘생산을 위한 여러 연구들에 의하면 양식산 뱀장어의 인공 성성숙 유도에 있어 외인성 호르몬의 투여 시기에 따라 투여 횟수에 상이한 차이를 나타내었고, 이러한 결과는 계절에 따라 생식소에 대한 GTH 반응성 차이에 의한 것으로 이러한 차이에 의해 번식률에 영향을 끼칠 것으로 추정된다. 따라서 계절별로 뱀장어의 인위적인 성성숙을 유도하였고 이에 따른 수정률, 부화율 및 자어의 생존율 등을 포함한 번식률을 조사하여 효과적인 인공 성성숙 유도를 위한 뱀장어 친어의 최적 성성숙 유도시기를 확인한 결과 자연상에서의 주 산란기간인 봄과 여름철(5~7월) 다른 계절들에 비해 번식률이 유의하게 높은 것으로 나타났으며, 상기 기간 동안에는 인공 성성숙 유도에 의해 자연산란 및 수정도 유도할 수 있었으며, 인공수정에 의해 획득된 수정란에 비해 번식률이 높은 것으로 나타났다. 이를 통해 뱀장어의 인공 성성숙 유도 시기는 봄과 여름철(5~7월) 적합할 것임을 제시하였다. 인공 성성숙 유도 후 자연산란 및 수정을 유도하여 획득한 수정란과 부화자어에 대한 형태학적 특징을 조사한 결과 뱀장어 수정란은 무색의 투명한 분리 부성란으로 난경은 평균 1 mm였으며, 전형적인 반상분할을 통해 난발생이 이루어졌으며, 수정 38시간 후부터 부화가 개시되었다. 부화 직후의 pre-leptocephalus 자어들은 전장 약 3 mm 정도의 조금 만곡한 상태로, 약 1시간 후부터 몸이 수평으로 향하였다. 부화 후 4.5일 후, 입과 항문이 형성되었고, 이 시기에 1차적으로 대량의 폐사가 발생하였다. 부화 후 7.5일 후, 소화기관과 가슴지느러미가 형성되기 시작하였다. 부화 후 8.5일 후, 난황흡수가 완료되었고, 소화기관이 발달되어 외부적으로 사료를 공급해 주었고, 자어는 사료 섭취를 개시하였다. 그러나 부화 후 14.5일 후 자어는 전량 폐사하였고, 이때 전장은 5.87±0.25 mm로 확인되었다. 대량폐사의 주요 원인은 자어에 적합한 사료를 공급해 주지 못한데 따른 사료 섭취율 저하와 이로 인해 난황 흡수 완료 후 영양결핍과 적절한 사육 시스템의 부재로 인해 폐사한 것으로 추정된다. 따라서 향후 부화자어의 사육을 위한 적합한 사료와 사육 시스템의 개발이 수반되어야 할 것으로 판단된다. 상기의 번식 내분비학적 연구 결과들에 의하면 뱀장어의 인공 종묘 생산에 있어 인공 성성숙 유도와 부화자어는 성공적으로 생산할 수 있었지만, 번식률과 부화자어의 생존율은 극히 미약한 것으로 나타났다. 따라서 본 연구에서는 번식 내분비학적 연구를 뒷받침하기 위한 연구의 일환으로 뱀장어의 번식률과 부화자어의 생존율 향상을 위한 사료 영양학적 연구를 통해 치어기 뱀장어의 주요 영양소 요구량을 구명하였다. 뱀장어 치어의 에너지에 대한 적정 단백질의 비율(P/E ratio)에 있 성장률과 사료효율 등을 고려한 에너지 수준별 사료 내 적정 단백질 함량은 에너지 19 kJ / g에서 단백질 함량 45% 실험구에서 최적의 성장을 나타내었으며, 이를 토대로 사료 내 적정 P/E ratio는 24 mg protein / kJ 가 적합한 것으로 판단된다. 필수지방산과 그 적정 요구량을 평가하기 위하여 n-3원으로써 α-lenolenic acid (LNA, 18:3n-3), docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3)와 n-6원으로써 linoleic acid (LA, 18:2n-6), arachidonic acid (ARA, 20:4n-6)을 사료 내 수준별로 첨가한 사료를 공급하여 성장과 체조성 및 혈액성상에 미치는 영향을 확인한 결과 뱀장어 치어의 필수지방산 요구량은 n-6 계열의 LA 과 n-3 계열의 LNA을 사료 내 1% 수준에서 1:1 혹은 1:2의 비율로 동시에 첨가해 주는 것이 적합한 것으로 나타났다. Arachidonic acid (ARA; 20:4n-6)의 적정 요구량을 평가하기 위해 사료 내 수준별로 첨가한 사료를 공급하여 사육실험을 수행하였고, 증체율과 사료효율에 대한 broken-line model 분석 결과, 뱀장어 치어의 성장과 사료효율 향상을 위한 사료 내 아라키돈산의 적정 첨가수준은 0.63% 보다는 높아야 하지만 0.71% 수준이면 충분할 것으로 확인되었다. 항산화 vitamin C (ascorbic acid)와 E (α-Tocopherol, TA)의 적정 요구량을 평가하기 위해 사료 내 각각의 수준별로 첨가한 사료를 공급하여 사육실험을 수행하였고, 증체율과 사료효율에 대한 broken-line model 분석결과, 뱀장어 치어의 성장과 사료효율 향상을 위한 사료 내 vitamin C의 적정 첨가수준은 24 mg/kg 보다는 높아야 하지만 38.1 mg/kg 수준이면 충분한 것으로 판단되고, vitamin E의 적정 첨가수준은 12.8 mg/kg보다는 높아야 하지만 16.5 mg/kg 수준이면 충분한 것으로 확인되었다. 상기의 주요 영양소 요구량에 대한 연구들은 부화자어와 친어의 영양학적 사육관리를 위한 기초자료를 제공하기 위한 목적으로 수행된 결과 성장 단계가 다른 치어기 뱀장어를 대상으로 평가되었다는 한계점이 있다. 이에 대해 실험적 규모로 뱀장어 친어를 대상으로 영양소의 요구량을 평가하기에는 한계가 있었고, 현 단계에서 사육실험을 위한 충분한 양의 부화자어를 확보할 수 있는 수준에 이르지 못하였다. 따라서 실험적 규모에 적합하고 확보와 관리가 용이한 치어기 뱀장어를 대상으로 주요 영양소 요구량을 구명하였고, 동일한 종에 있어 성장단계가 진행됨에 따라 영양소의 요구량이 감소하는 경향이 있음을 감안한다면, 향후 부화자어의 적합한 사료 개발을 통한 생존율 향상과 친어의 영양학적 관리를 통한 번식률 향상에 기초자료를 활용할 수 있을 것으로 판단된다.

Series of studies were conducted to induce sexual maturation for the artificial propagation of eel, Anguilla japonica and to evaluate major nutrients requirements of juvenile eel for the managements of hatched larvae and broodstock by nutritional approaches. Firstly, five experiments were examined for sex determination of cultured eel for broodstock managements (Exp. 1-1), induction of artificially sexual maturation in male eel (Exp. 1-2), induction of artificially sexual maturation in male eel (Exp. 1-3), development of the eggs and pre-leptocephalus larvae by natural spawning of artificially matured eel (Exp. 1-4), and determination of optimum induction periods for artificially sexual maturation in cultured eel (Exp. 1-5). Secondly, five experiments were conducted to evaluate optimum dietary protein to energy ratio (Exp. 2-1) and requirements of optimum dietary essential fatty acids (Exp. 2-2), arachidonic acid (Exp. 2-3), antioxidant vitamins (Exp. 2-4) for juvenile eel, A. japonica. 1. Induction of artificial maturation for the seedling of eel, broodstock Experiment 1-1. Methods of sex determination for broodstock managements in cultured eel This study investigated sex determination through serum steroid hormone level and compared hematological characteristics of one-year old cultured eels, Anguilla japonica. Four different groups were divided with total length at 40~45, 45~50, 50~55 and 55~60 cm. Males dominated eels of 40~50 cm in total length group, while females dominated eels of more than 50 cm. All females and males were immature. Hematocrit levels of males (total length: 40~50 cm) were higher than those of females (total length: 50~60 cm). Hemoglobin content also differed between males and females. Glutamate oxaloacetate transminase (GOT) levels of 40~45 cm males tended to be higher (157±3.46 IU/L) than that of other sized males, but there were no significant differences between groups (P<0.05). The highest GOT levels were found in of 55~60cm females (148±3.46 IU/L) compared with that of other female groups (P<0.05). Glutamate pyruvate transaminase (GPT) levels of males and females in the 55~60cm size range was significantly higher than others (P<0.05). The result of this study indicated steroid hormone content of males and females were very low, but testosterone (T) and estradiol-17β (E2)contents of females were higher than those of males. For further research of sex determination in cultured eels, it needs to investigate in more defined body length. Experiment 1-2. Induction of artificial maturation in male eel This study was conducted to investigate the changes in body weight (BW), plasma sex steroid hormone profiles and testicular developments of cultured male eel Anguilla japonica during artificial maturation process. Eels that received weekly intraperitoneal injections of eel’s ringer solution containing human chronic gonadotropin (HCG) were examined. In ringer-treated control, the BW changes decreased slowly during the experiment period. Plasma testosterone (T), 11-ketotestosterone (11-KT) and 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) levels in control remained low and did not show any significant changes. Moreover, all germ cells in the testes of the control were spermatogonia. In the HCG-treated male eels, however, the BW changes gradually increased from 5th week and then slowly decreased. Plasma T level rapidly increased (P<0.05) at 2nd week and then slowly decreased. Plasma 11-KT level dramatically increased (P<0.05) at 2nd week and maintained until the end of the experiment. Plasma DHP level progressively increased from 2nd week and peaked at 8th week (P<0.05). The HCG-treated male eels had more developed testes; most were at spermatozoa and spermatid stages and showed active spermiation. Thus, spermatogenesis and spermiation in the cultured eel can be induced by repeated injections of HCG. Experiment 1-3. Induction of artificial maturation in female eel The present study demonstrates the changes in body weight (BW) and plasma sex steroid hormone profiles during artificial maturation induced by human chorionic gonadotropin (HCG) or salmon pituitary extract (SPE) injections in cultured eel Anguilla japonica, kept in seawater for 3 months. In the weekly SPE-injected female group, BW was relatively stable during vitellogenesis. Following induction of vitellogenesis, females exhibited a rapid increase of BW, and the oocytes were observed to be in the migratory nucleus stage at the end of the experiment. Plasma testosterone (T) and estradiol-17β (E2) levels increased slightly during vitellogenesis and peaked at an average of 5.82 ng/mL and 4.76 ng/mL, respectively, at the end of the experiment. In the weekly control and HCG-injected female groups, BW slowly decreased during the experimental period, and the oocytes of the two groups were observed to be at the primary yolk globule stage. In the weekly HCG injected female group, plasma T and E2 levels increased slightly during vitellogenesis and decreased afterward. In the control female group, however, plasma T and E2 levels were not altered during the experimental period. Furthermore, plasma 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) was not detected in all experimental groups. Fertility and hatching rates of SPE-injected females were significantly higher in those that ovulated 15 h after DHP injection than 18 h. These results indicate that long rearing in seawater increases responsiveness to SPE in ovarian maturation of the eel A. japonica, resulting in shortened period from completion of vitellogenesis by sex steroid hormone production. Experiment 1-4. Development of the eggs and pre-leptocephalus larvae by natural spawning of artificially matured eel Embryonic and pre-leptocephalic larvae development of the eel, Anguilla japonica, are described following natural fertilization in the indoor tank of 23℃ water temperature. Following a routine hormone treatment technique for the brood stock, female eels were artificially matured by weekly intramuscular injections of salmon pituitary extracts (SPE) at a dosage of 20 mg/kg body weight (BW) for a total of 10-14 doses to induce ovarian maturation, while male eels received weekly intramuscular injections of human chorionic gonadotropin (HCG) at a dosage of 1 IU/g BW for a total of 6-10 doses to induce testicular maturation in a separate aquarium and induced natural spawning. Fertilized eggs of about 1.0 mm in diameter were pelagic and showed a typical discoidal cleavage. Hatching occurs 38 hrs after fertilization at a water temperature of 23℃. The newly hatched larvae measured about 3.0 mm in total length and the number of myomeres averages 42. Their mouths and anuses were opened at 4.5 days and the yolk sacs of the pre-leptocephalic larvae were almost absorbed at 6.5 days after hatching. Preleptocephalic larvae survive for 14.5 days. At this time they are 5.87±0.25 mm in total length and have about 98 myomeres. However, morphological characterization of embryonic and pre-leptocephalic larvae were not different between natural fertilization and artificial fertilization by the dry method. Experiment 1-5. Determination of optimum induction periods for artificially sexual maturation in cultured eel This study investigated the correlationship between artificial maturation season and reproduction coefficient of cultured eel Anguilla japonica from May (spring) to next January (winter). The brood stock, female eels (400~600 g) were artificially matured by weekly intramuscular injections of salmon pituitary extracts (SPE, 20 mg/fish) to induce a completion of vitellogenesis. After completion of vitellogenesis, final oocyte maturation and ovulation was induced by injection of 17α, 20β-dihydroxyprogesterone (DHP) at about 2 μg/g body weight. Most fish ovulated 15~18 h following the DHP injection. The ovulated fish were induced to natural spawning or artificial fertilization by the dry method. Males (200~350 g) were received weekly intramuscular injections of human chorionic gonadotropin (HCG) at a dosage of 1 IU/g body weight to induce testicular maturation and spermiation. Seasonal reproduction coefficient which includes the rate of ovulation, buoyancy, fertilization and hatching of eggs in the artificially matured eel during spring to summer (May~July) were significantly higher than the other season, while there were no significant difference among spring and summer (P<0.05). Furthermore, the number of eggs spawned and larvae hatched in the artificially maturated eel during spring to summer (May~July) were significantly higher than the other season, while there were no significant difference in spring and summer (P<0.05). These results indicate that artificial maturation by hormone treatment of A. japonica was successful only during spring to summer, which is the maturation period in the wild stock in nature. Consequently, it is possible to determine the period of artificially induced sexual maturity by the reproduction coefficient which includes the rate of ovulation, buoyancy, fertilization and hatching of eggs in the cultured eel A. japonica. 2. Nutritional studies of juvenile eel Experiment 2-1. Evaluation of the optimum dietary protein to energy ratio A 16-week feeding trial was conducted to estimate the optimum dietary protein to energy ratio (P/E ratio, mg protein/kJ) in juvenile Japanese eel, Anguilla japonica. Six experimental diets were formulated with three energy levels and two protein levels at each energy level. Three energy levels of 16, 17 and 19 kJ per g diets were included at 45 and 50% crude protein (CP) levels, respectively (45P16, 45P17, 45P19, 50P17, 50P17 and 50P19). After four weeks of the conditioning period, fish initially averaging 15±0.3 g (means±SD) were randomly distributed into each tank as groups of 20 fish. Each diet was fed to fish in three randomly selected tanks at a rate of 2-3% wet body weight per day in the recirculated system. Weight gain (WG) and specific growth rate of fish fed diet 45P19 were significantly higher (P<0.05) than those of fish fed the other diets. WG of fish fed diet 50P17 was also significantly higher than those of fish fed diets 50P16 and 50P19. Feed efficiency ratio of fish fed diets 45P19 was significantly higher (P<0.05) than those of fish fed other diets, however 45P17 and 45P19 were no significant differences (P>0.05). These results suggest that the optimum P/E ratio may be 24.1 mg protein/kJ with 44.3% protein diets, and 28.1 mg protein/kJ 49.3% protein diets for the maximum growth of juvenile eel under the experimental condition. Experiment 2-2. Evaluation of the optimum dietary essential fatty acids requirements The present study was conducted to evaluate the optimum dietary requirements of essential fatty acids (EFAs) such as linoleic acid (LA, 18:2n-6), α-lenolenic acid (LNA, 18:3n-3), or docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA, 20:4n-6) in juvenile eel Anguilla japonica cultured in the recirculating system for 16 weeks. The experimental diets contained 50% crude protein 10% crude lipid and 3800 kcal/kg energy. Brown fish meal and blood meal were used as the main protein sources, while coconut oil, corn oil and linseed oil were used as the lipid source to get different fatty acids ratios. The effects of the essential fatty acids supplementation on weight gain (WG), specific growth rate (SGR), feeding efficiency (FE), proximate composition and fatty acids contents of whole body were examined after the feeding trial. WG, SGR, and FE of fish fed diet D2, D3 were significantly higher (P<0.05) than those of fish fed the other diets. HUFA concentration of whole body of fish fed D1 was significantly lower (P<0.05) than those of fish fed the other diets. HUFA/SFA (saturated fatty acids) ratio of whole body in fish fed diets D2, D3 and D6 were significantly higher than that of fish fed diet D1 (P<0.05). DHA/EPA ratio of whole body in fish fed diet D7 was significantly higher than those of fish fed the other diets; and fish fed diet D5 showed the lowest DHA/EPA ratio among all the dietary treatments (P<0.05). Based on the experimental results, we concluded that LNA (n-3) and LA (n-6) were necessary for optimum growth of juvenile eel, and the dietary requirement of LNA and LA were 0.35-0.5% and 0.5-0.65%, respectively. Experiment 2-3. Evaluation of the optimum dietary arachidonic acid requirements and essentiality This study was conducted to evaluate the optimum dietary arachidonic acid (ARA) requirements on growth performance, body composition and fatty acid profile of juvenile eel, Anguilla japonica. Six experimental diets (18.3 kJ of available energy g-1) were formulated to be iso-nitrogenous and iso-caloric to contain 55.0% crude protein, 15% crude lipid. Six different levels of ARA were added to the basal diet, with 0, 0.2, 0.4, 0.6, 0.8 and 1.2% per kg diet on dry matter (DM) basis, respectively (ARA0, ARA0.2, ARA0.4, ARA0.6, ARA0.8 and ARA1.2). After 2 conditioning period, fish initially averaging 27±0.5 g (mean±SD) were randomly distributed into each aquarium as triplicate groups of 20 fish. Water temperature was maintained at 25±1 ℃. One of six experimental diets was fed on a DM basis to fish in three randomly selected aquaria at rate of 3-4% of total body weight twice a day. Growth performance and ARA deposition of whole body in fish fed ARA0.6 and ARA0.8 diets were significantly higher than that of the other diets (P<0.05), there were no significant differences among fish fed ARA0.6 and ARA0.8 diets. Broken-line model analyses of WG and FE were 0.63 and 0.71 % in diet, respectively. These results indicated that the optimum dietary arachidonic acid levels could be greater than 0.63% but less than 0.71% in juvenile eel. Experiment 2-4. Evaluation of the optimum dietary antioxidant vitamin C and E requirements This study was conducted to evaluate the optimum dietary antioxidant vitamin C (ascorbic acid, AA) and vitamin E (α-tocopherol, TA) requirements on growth performance and body composition of juvenile eel, Anguilla japonica. Five different levels of AA were added to the basal diet, with 0, 30, 60, 120 and 1200 mg AA per kg diet on dry matter (DM) basis, respectively (AA0, AA30, AA60, AA120 and AA1200). Five different levels of TA were added to the basal diet, with 0, 15, 30, 60 and 120 mg TA per kg diet on DM basis, respectively (TA0, TA15, TA30, TA60 and TA120). After 2 conditioning period, fish initially averaging 15±0.3 g (mean±SD) were randomly distributed into each aquarium as triplicate groups of 20 fish. Water temperature was maintained at 25±1℃. One of five experimental diets was fed on a DM basis to fish in three randomly selected aquaria at rate of 3-4% of total body weight twice a day. Broken-line model analyses of WG was 38.1 mg/kg in diet, respectively. These results indicated that the optimum dietary vitamin C levels could be greater than 24 mg/kg but less than 38.1 mg/kg diet in juvenile eel. Broken-line model analyses of WG was 12.8 mg/kg in diet, respectively. These results indicated that the optimum dietary vitamin E levels could be greater than 12.8 mg/kg but less than 16.5 mg/kg diet in juvenile eel.