666 윤주연 허성범 재료및방법 Brachionus plicatilis 의내구란생산 rotifer (Culture Collection of Useful Marine Plankton, CCUMP) B. plicatilis (CCUMP 41) strain. rotifer

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1 Kor J Fish Aquat Sci 44(6), DOI : 44(6), , 2011 보관방법에따른 Rotifer Brachionus plicatilis 내구란의부화 윤주연 허성범 부경대학교해양바이오신소재학과 / 한국해양미세조류은행 The Hatching Rate of Resting Eggs of the Rotifer Brachionus plicatilis according to Preservation Method Joo-Yeon Youn and Sung Bum Hur Korea Marine Microalgae Culture Center / Department of Marine Bio-materials and Aquaculture, Pukyong National University, Busan , Korea The rotifer Brachionus plicatilis is one of the most important food organisms in aquaculture. The resting eggs produced by mictic female rotifers are easily stored and hatched, making them useful as the starter for the mass culture of rotifers in marine larval culture. This study examined the optimum preservation method for resting eggs to ensure a high hatching rate. To produce resting eggs, the marine rotifer B. plicatilis was cultured with Nannochloris oculata (KMMCC 16). The resting eggs were harvested and cryopreserved using 5% and 10% methanol (MeOH), dimethylsulfoxide (DMSO), and glycerol as cryoprotectant agents (CPAs). The cryopreservation comprised slow or rapid freezing and the resting eggs were stored for one month in liquid nitrogen (-196 C). The resting eggs were also dried at different temperatures (30, 40, and 50 C) and for different times (1, 2, and 3 h). In general, the hatching rates of the resting eggs preserved with CPA were higher than those without CPA and the slow freezing method was better than the rapid freezing method. However, the optimum CPA concentration for the hatching rate of the resting eggs varied with the freezing method and kind of CPA, and the CPA also affected the viility of the resting eggs. Dried resting eggs had a high, rapid hatching rate over 80%. The moisture content of the resting eggs cryopreserved in liquid nitrogen affected the hatching rate. Drying at 30 C for 1 hour resulted in a high hatching rate of the resting eggs. In conclusion, drying at 30 C for 1 hour and preservation in liquid nitrogen with the slow freezing method, without CPA, is recommended for a high hatching rate (ca. 95%) of rotifer resting eggs. Key words: Brachionus plicatilis, Cryopreservation, Dry, Hatching rate, Resting eggs 서 론 Rotifer Brachionus plicatilis,. (Lubzens, 1987, Planas et al., 2004). rotifer,, ph, COD (Yu and Hirayama, 1986) rotifer (Hayshi et al., 2007). rotifer. Rotifer, *Corresponding author: hurs@pknu.ac.kr (Yamasaki and Hirata, 1982) (Lubzens et al.,1990, Zhou et al., 2001). rotifer (B. plicatilis) (B. rotundiformis) 4 (Hagiwara et al., 1997). Rotifer Artemia cyst (Hagiwara et al., 1997, Zhou et al., 2001).. (Park and Hur, 1996a,b). B. plicatilis. 665

2 666 윤주연 허성범 재료및방법 Brachionus plicatilis 의내구란생산 rotifer (Culture Collection of Useful Marine Plankton, CCUMP) B. plicatilis (CCUMP 41) strain. rotifer 5 L 3 L 28, 25 psu 50 µmolm -2 s -1. Rotifer (Korea Marine Microalgae Culture Center, KMMCC) Nannochloris oculeata (KMMCC 16) f/2 (Guillard and Ryther, 1962) (3,000 rpm, 25 min). Rotifer /ml µm seive 30 ml test tube 4. 보관방법 Rotifer (LN 2 ). (cryopreotectant agents, CPAs) MeOH (methanol), DMSO (dimethyl sulphoxide, Me 2 SO) glycerol. MeOH DMSO 0.45 µm G/F glycerol. cryovial 1.5 ml 25 psu 50 CPAs 5% 10% , 1 1 Mr. Frosty Freezing Container ( , Nalgene) (Gwo et al., 2005). isopropanol 200 ml Freezing Container CPAs CPAs incubator Mr. Frosty Freezing CPAs 5% 10% MeOH.. Fig 1. Daily variations of hatching rate of the resting eggs of Brachionus plicatilis preserved by slow freezing method with CPAs (up), rapid cooling method with CPAs (middle) and dry method (bottom) at 20 and 25 psu for ten days. (CPA : cryoprotect agent, LN 2 : liquid nitrogen). 35 water bath , 25 psu, 50 µmolm -2 s -1 3 ml multi-culture plate 1

Rotifer 667 Fig 2. Final hatching rate of the resting eggs of Brachionus plicatilis preserved by different cryopreservation methods. The resting eggs were hatched out at 20 and 25 psu for ten days.

3 Rotifer 667 Fig 2. Final hatching rate of the resting eggs of Brachionus plicatilis preserved by different cryopreservation methods. The resting eggs were hatched out at 20 and 25 psu for ten days. A different letter on the bar means a significant difference at P<0.05 level. (CPA : cryoprotect agent, LN 2 : liquid nitrogen). 10 rotifer. 3. 건조온도와시간에따른내구란의부화실험. 50 petri dish. petri dish 30, , 2, desicator ml multi-culture plate 1 25 psu 24, 50 µmolm -2 s , , 2, 3 2. 통계분석 SPSS (SPSS Inc. version 17.0) one-way ANOVA-test Duncan s multiple range test (Duncan, 1955) (P<0.05). 결 보관방법에따른부화율 과 B. plicatilis Fig. 1, 2. Freezing Container 5% MeOH 2 80% 6 83%. 10% MeOH 2 44%, 10 61% 5%. 5% DMSO 2 10% 7 35%, DMSO 10% 7 17% 5%. Glycerol 5% 3 4% 9 28%, 10% 2 15%, 9 45%.

4 668 윤주연 허성범 Decreasing rate of moisture content (%) a a b c d Drying Hours 1 hr 2 hr 3 hr 30 C 40 C 50 C % MeOH 83% DMSO glycerol (P<0.05). MeOH DMSO CPAs glycerol. 5% MeOH 10 8% 10% 2 39% 9 46% (P<0.05). DMSO 5% 2 21% 6 59% 10%. Glycerol 5% 10 36% 10% e Temperature ( C) f g h Drying Hours 1 hr 2 hr 3 hr Fig 3. Decreasing rate of moisture content (up) of the resting eggs of Brachionus plicatilis resting eggs dried at different temperatures and hours and their hatching rate (bottom) at 24 and 25 psu for ten days. A different letter on the bar means a significant difference at P<0.05 level. c bc cd 50 i d 2 3%, 8 17%. CPAs 10 8% (P<0.05) %, 5 30% 6 84%, 8 89%. CPAs 10 95% % MeOH 10 81%, 10% MeOH 4 83% 5% MeOH. 건조온도와시간에따른내구란의수분함량감소율및부화율 Fig % 2, 3 81% 79%. 40 1, 2, 3 59%, 57% 52%, 50 38%, 33%, 31% (P<0.05) %, 2 86%, 3 85% % % (P<0.05). 고 찰 living organism,, (Poncet and Veron, 2003), (Rhodes et al., 2006),. (Canavate and Lubian, 1995; Youn and Hur, 2009). Rotifer 2% (King et al.,1983). rotifer amictic female egg cleavage, invaginatiat, symmetrical embrya, eyed 4 10% DMSO (-196 ),

5 Rotifer % (Toledo and Kurokura,1990). Artemia cyst (Hagiwara and Hino, 1989, Kogane et al., 1997) L:24D 25 40%., 6 (Hagiwara and Hino, 1989). Park and Hur (1996b) rotifer 28, 40 µmolm - 2 s %, % 40%., rotifer (Balompapueng et al., 1997). CPA 5% 10% CPA CPA. DMSO. CPA CPA. 5% MeOH 2 80%, 6 83% %, 95%. 20. (Park and Hur, 1996b), % 92%, % 74%. Park and Hur (1996b) 30 1 (46.4%) % 79% 50 38% 31% % CPA. CPA CPA rotifer CPA. 사 사 2011 ( ) 참고문헌 Balompapueng MD, Hagiwara A, Nozaki Y and Hirayama K Preservation of resting eggs of the euryhaline rotifer Brachionus plicatilis O. F. Müller by canning. Hydrobiologia 358, Canavate JP and Lubian LM Relationship between cooling rate, cryoprotectant concentrations and salinities in the cryopreservation of marine microalgae. Mar Biol 124, Duncan DB Multiple-range and multiple F test. Biometrics 11, Guillard RRL and Ryther JH Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Can J Microbiol 8, Gwo JC, Chiu JY, Chou CC and Cheng HY Cryopreservation of a marine microalga, Nannochloropsis oculata (Eustigmatophyceae). Cryobiology 50, Hagiwara A, Balompapueng MD, Munuswamy N and Kirayama Mass production and preservation of the resting eggs of the euryhaline rotifer Brachionus plicatilis and B. rotundiformis. Aquaculutre 155, Hagiwara A and Hino A Effect of incubation and preservation on resting egg hatching and mixis in the derived clones of the rotifer Brachionus plicatilis. Hydrobiologia 186/187, Hayashi M, Yukino T, Watane F, Miyamoto E and Nakano Y Effect of vitamin B12-enriched Thraustochytrids on

6 670 윤주연 허성범 the population growth of rotifers. Biosci Biotechnol Biochem 71, King CE, Bayne Bayne H, Cannon TK and King AE Cryopreservation o f monogonont rotifers. Hydrobiologia 104, Kogane T, Hagiwara A. and Imaizumi K Temperature conditions enhancing resting egg production of the euryhaline rotifer Brachionus plicatilis O. F. Muller (Kamiura strain). Hydrobiologia 358, Lubzens E Raising rotifers for use in aquaculture. Hydrobiologia 147, Lubzens E, Kolodny G, Perry B, Galai N, Sheshinski R and Wax Y Factors affecting survival of rotifers (Brachionus plicatilis O.F. Müller) at 4. Aquaculture 91, Park HG and Hur SB. 1996a. Production and hatching rate of resting egg of Korean rotifer, Brachionus plicatilis (S-type) with different diets. J Aquaculture 9, Park HG and Hur SB. 1996b. Effect of temperature, salinity and preservation method on hatching rate of resting egg of Korean rotifer, Brachionus plicatilis (S-type). J Aquaculture 9, Planas M, Vázquez JA, Marqués J, Pérez-Lomba R, Gonzáles MP and Murado M Enhancement of rotifer (Brachionus plicatilis) growth by using terrestrial lactic acid bactera. Aquaculture 240, Poncet JM and Veron B Cryopreservation of the unicellular marine alga, Nannochloropsis oculate. Biotechnology Letters 25, Rhodes L, Smith J, Tervit R, Roberts R, Adamson J, Adams S and Decker M Cryoperservation of economically valule marine micro-algae in the classes Bacillariophyceae, Chlorophyceae, Cyanophyceae, Dinophyceae, Haptophyceae, Prasinophyceae, and Rhodophyceae. Cryobiology 52, Toledo JD and Kurokura H Cryopreservation of the euryhaline rotifer Brachionus plicatilis embryos. Aquaculture 91, Yamasaki S and Hirata H Rearing of the prawn, Penaeus japonicus, fed on frozen and living rotifers. Min Rev Data File Fish Res 2, Youn JY and Hur SB Cryopreserved marine microalgae grown using different freezing methods. Algae 24, Yu J and Hirayama K The effect of un-ionized ammonia on the population growth of the rotifer in mass culutre. Nippon Suisan Gakkaishi 52, Zhou L, Zheng Y and Xiang JH Study on mixis potential of rotifer rseting eggs (Brachionus plicatilis) with different collection times and different preservation periods. Chinese J Oceanol Limnol 19,

19(4)-07(43).fm

19(4)-07(43).fm J. of Aquaculture Vol. 19(4) : 310-314, 2006 µ Journal of Aquaculture Korean Aquaculture Society» cyclopoid ƒ Paracyclopina nana,» 1, x * w w, 1 w w œw Grazing of Brackish Water Cyclopoid Copepod Paracyclopina