Cryopreservation of the sea perch ( L ateolabrax japonicus) embryos by vitrif ication 3

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1 49 (6) : A cta Zoologica S inica 3 33 ( ) ( ) ( L ateolabrax japonicus) %100 % %6310 % ; VSD2 20 VSD2 : 015 mol/ L 1020 min VSD2 ( ) 211 %2719 % h ; 1 [ 49 (6) : ] Cryopreservation of the sea perch ( L ateolabrax japonicus) embryos by vitrif ication 3 TIAN Yong2Sheng CHEN Song2Lin 33 YAN An2Sheng J I Xiang2Shan YU Guo2Cai ( Huaz hong A gricult ural U niversity W uhan Chi na) ( Yeallow Sea Fishery Research Instit ute CA FS Key L ab For S ustai nable Utilization of Mari ne Fisheries Resources Mi nist ry of A gricult ure Qi ngdao S handong China) Abstract To improve cryopreservtion of sea perch ( L ateolabrax japonicus) embryos we selected five vitrification solu2 tions that could vitrify consistently. The cryoprotectants concentration in the five vitrification solutions were low to reduce toxicity. The vitrification probability of the five solutions was 4811 % % during freezing and 4414 % % dur2 ing thawing at water bath respectively. VSD2 was more stable during thawing than the other four vitrification solutions and was used to cryopreserve sea perch embryos in the experiment. The durable time of sea perch neurula stage 202pair2muscle stage tail2bud stage heart2beating stage and prehatching stage in VSD2 was compared and the most suit2 able stage to cryopreserve by vitrification was selected. We found that the neurula stage sustained the least time in VSD2 and the heart2beating stage was the most suitable stage to cryopreserve by vitrification. The pre2hatching stage was also feasible to cryopreserve by vitrification. The effect of different elution times in 015 mol/ L sucrose was compared and elu2 tion for 10-20min in 015 mol/ L sucrose was found to be efficient. We used VSD2 as vitrification solution to cryopreserve ( ) sea perch embryos of different stages and got 211 % % transparent embryos after thawing. Further2 more two frozen2thawed embryos at heart2beating survived h after thawing. Another frozen2thawed embryo at pre2hatching stage hatched [ Acta Zoologica Sinica 49 (6) : ]. Key words Sea perch ( L ateolabrax japonicus) Embryos Vitrification Cryopreservation ( 2002) (Vitrification) (No : 2001AA621100) [ This research was fund2 ed by a grant from State 863 High2technology R &D Project of China (No. 2001AA621100) ] 33 (Corresponding author). E2mail : ac. cn 38 : ν 2003 Acta Zoologica Sinica

2 ( 1998) (Rall et al. 1985) (Massip et al ; Douchi et al. 1990) (Szell et al. 1989) ( Kasai et al. 1992) (Zhu et al. 1993) Rall et al. (1985) 111 VS 1 : 2015 %DMSO % + 10 % % ( PEG: ) L HRH2A g/ kghcg EFS40 ( Kasai et al ; Zhu et al. 1993) 100 IU/ kg 35 g/ kg L HRH2A IU/ kg HCG % + 20 % ( 2002) ( PG) ( MeOH) ( Gly) ( EG) (DMF) (DMSO) NaCIKCID2 ( Glucose) (Sucrose) ( Fahy 1981) 113 PGMeOHGly DMFEGDMSO PG < MeOH < Gly < DMF < EG < DMSO PG 15 %20 %25 %30 % ( v/ v) 4 D15 ( 1992) ( 2002) A B CD4 80 : min ( Zhang et al. 1989) ( 1997) 35 % h ( 2002) kg VSD2 ( L ateolabrax japonicus ) ( Perciforms ) ( Servanidae) (Lateo2 ( labrax) )

3 6 : 4 VSD2 1/ 4 1/ 3 1/ 2 2/ 3 1 VSD2 015 mol/ L 10 min 1417 Table 1 Vitrif ication solutions VSD2 and tha wing temperatures ( n = 27) 115 VSD min (1515 ) 015 mol/ L 2 ml min 2 ml VSD2 4 VSD2 250 l VSD min 1 ; VSD mol/ L 2 ml 20 min 815 % 40 min 10 min 1 min 2 ml 3 1 h 20 VSD2 20 ; min 8313 % / %1719 %10163 % VSD VSD2 20 min 3813 % ( P > 0105) ( P < 0105) VSD Code 1 ( n = 27) Component ( %) Vitrification degree ( ) Thawing temperature Cryopreservation Thawing ( ) VSB16 20 %PG + 30 %EG VSC3 25 %PG + 25 %MeOH VSD2 30 %PG + 20 %MeOH VSD10 30 %PG + 20 %DMF VSD14 30 %PG + 20 %EG min 9318 % 4211 %4715 %10163 % 211 VSD min 9518 % %7313 %6112 %8144 % 1175 % 1 1 VSD2 50 min % 4811 %100 % min 4414 % VSD %VSD % min 8915 % 6310 % 7718 %7116 %3012 %915 % 70 min VSB VSD2 ( ) VSD min 845

4 VSD2 ( n = 3) Fig. 1 The varying curve of mean survival rates of sea perch embryos in different stages in VSD2 VSD2 min10 min15 min20 min VSD2 ( P < 0105) 520 min ( P > 0105) VSD2 2 2 VSD2 Table 2 The optimal equilibration time of sea perch embryos in different stages in VSD2 (min) ( %) Optimal equilibration Stage of embryos Survival rate time (Neurula) < 20 < (20 pairs of muscle stage) ( Tail2bud stage) ( Heart2beating stage) ( Pre2hatching stage) mol/ L min 5517 %6419 %7019 % 2 20 Fig. 2 Survival rate of sea perch 20 pairs of muscle stage in different elution time (Different letters indicate significant dif2 ference One2Way ANOVA LSD test P < 0105) 214 VSD2 30 min min 6177 % ( 3) ; 20 VSD min min % 20 VSD2 30 min 211 %1915 %1312 % 1311 % ; VSD min h 6519 %2915 % 15 min 100 %97198 %100 % (2) : 25 min 5

5 6 : 3 Table 3 Results of vitrif icable cryopreservation of sea perch embryos ( %) ( %) (min) (min) Percentage Percentage of ( %) (h) Stage of Equilibrait Freezing Total Numbers of of intact transparent Survival rate Alive time embryos on time time specimens alive embryos embryos embryos (Neurula) 20 (20 pairs of muscle stage) ( Tail2bud stage) ( Heart2beating stage) ( Pre2hatching stage) %6104 %1316 % ; VSD min min 9117 %8418 % 8718 % 1118 %815 %2719 % VSD min 1 ( H30 H40) 5188 % 2113 % H30 H40 (: A B) H30 H40 30 h VSD (: C D) H40 42 h H30 42 h 2113 %5188 % 312 VSD2 50 h VSD min min 8212 %8816 %100 % 9015 % VSD2 70 min 1 ( 49 h (: F) (Zhang et al. 1995) ( 1994) %60 % (Robertson et al. 1988) ( Ctenopharyngo2 don idell us) DMSO P70 : E) 4176 % DMSO ( 1992) ( B rachydanio rerio)

6 h VSD2. :. ] VSD mol/ L 525 min 15 min 1020 min 314 VSD %2719 % h VSD ( References) Chen S. L Progress and prospect of cryopreservation of fish gametes and embryos. Journal of Fisheries of China 26 (2) : 161 Zhang L. Z. D. C. Lu S. L. Chen and J. P. Fang 1992 Ef [ 2002 fects of several factors on the survival rate of fish embtyos before. 26 (2) : ] cryopreservation. Freshw ater Fisheries 1 : [ Chen S. L. X. T. Liu D. C. Lu L. Z. Zhang C. J. Fu and J P. Fang 1992 Cryopreservation of spermatozoa of silver carp common carp blunt snout bream and grass carp. Acta Zool. Sin. 38 (4) : [ (4) : ] Douchi O. H. Takakura and K. Imai 1990 Transfer of bovine Fahy G. embryos cryopreserved by vitrification. J. A ni m. Reprod. 36 (1) : M Prospects for vitrification of whole organs. Cryobiology 18 : 617. Hua Z. Z. and H. S. Ren 1994 Cryobiological Medical Technolo2 gy. Beijing : Science Press. [ 1994 Kasai M. J. H. Komi A. Takakamo H. Tsudera T. Sakurai and T. Machida 1990 A simple method for mouse embry cryop2 reservation in a low toxicity vitrification solution without apprecia2 ble loss of viability. J. Reprod. Fertil. 89 : Kasai M. Y. Hamaguchi S. E. Zhu T. Sakurai and T. Machida 1992 High survival of rabbit morulae ofter vitrification in an ethy2 lene glycol based solution by a simple method. Biol. Reprod. 46 : Li G. W. C. Y. Zheng and B. Tang 1998 Cryobiology. Chang2 sha : Hunan Science and Technology Press. [ :. ] Massip A. P. Van Der Zwalman B. Scheffen and F. Ecotors 1986 Pregnancies following transfer of cattle embryos preserved by vitrification. Cryo2letters 7 : Rall W. F. and G. M. Fahy 1985 Ice2free cryopreservation of mouse embryos at by vitrification. N at ure 313 : Robertson S. M. A. L. Lawrence W. H. Nell C. R. Arnold and G. Mccarty 1988 Toxicity of the cryoprotectants glycerol dimethyl sulfoxide ethylene glycol methanol sucrose and sea salt solutions to the embryos of red drum. t urist. 50 : Progressive Fish2Cul2 Szell A. J. N. Shelton and K. Szell 1989 Osmotic chacteristics of sheep and cattle embryos. Cryobiology 26 : Zhang K. J. Y. D. Lou Y. J. Zhang and P. Wu 1997 Stud2 ies on cryopreservation of three freshwater fishes embryos. Journal of Fisheries of Chi na 21 (4) : [ (4) : ] Zhang L. Z. D. C. Lu L. Liu F. Guo and J. M. Zhang 2002 Research on cryopreservation of oriental weather fish embryo with vitrification solutions ( Misgum us anguillicaudat us). Journal of Fisheries of Chi na 26 (3) : [ (3) : ]. 1 : ] Zhang T. and D. M. Rawson 1995 Studies on chilling sensitivity of zebrafish ( B rachydanio rerio) embryos. Cryobiology 32 (3) : Zhang X. S. L. Zhao T. C. Hua and H. Y. Zhu 1989 A study on the cryopreservation of common carp Cyprinus carpio em2

7 6 : bryos. Cryo Letters 10 : Zhu S. E. M. Kasai H. Otoge T. Sakurai and T. Machida 1993 Cryopreservation of expanded mouse blastocysts by vitrifi2 cation in ethylene glycol2basad solutions. J. Reprod. Fertil. 98 : ( Explanation of Plate) ( Plate ) A. H30 : VSD2 30 min min ( H30 : the survived heart beating stage after elution. It was equilibrated for 30 min in VSD2 and frozen for 29 min in liquid nitrogen. The surface of the heart beating stage was coarser than that of the natural embryo) 60 B. H40 : VSD2 40 min min ( H40 : the survived heart beating stage after elution. It was equilibrated for 40 min in VSD2 and frozen for 58 min in liquid nitrogen. The tail of embryo swelled) 60 C. H30 : 30 h 50 h ( H30 : the hatched heart beating stage after culture for 30 h. It died after culture for 50 h and was not significantly different from natural embryos) 60 D. H40 : 30 h 42 h ( H40 : the hatching stage after culture for 30 h. It died after culture for 42 h) 60 E. H70 : VSD2 70 min min ( H70 : the survived pre2hatching stage after elu2 tion. It was equilibrated for 70 min in VSD2 and frozen for 50 min in liquid nitrogen) 60 F. H70 : 59 h ( H70 : hatched fry. It survived for 59 h and the tail curled) 60

8 : TIAN Yong2Sheng et al. : Cryopreservation of t he sea perch ( L ateolabrax japonicus) embryos by vitrification Plate ( Explanation at the end of the text)

Design of vitrification solutions for the cryopreservation of embryos

Design of vitrification solutions for the cryopreservation of embryos J. Ali and J. N. Shelton Developmental Physiology Group, fohn Curtin School of Medical Research, Australian National University, Canberra,