34 6 () V o l. 34 N o. 6 2006 6 Jour. of N o rthw est Sci2T ech U niv. of A gri. and Fo r. (N aṫ Sci. Ed. ) Jun. 2006 Ξ 1, 1, 2, 1, 1, 1, 1, 1 (1, 712100; 2, 066004) [ ], 3, 0. 035 ggml,, (46. 34 0. 52) %, (41. 38 0. 39) % (43. 51 1. 78) %, (P > 0. 05), (P < 0. 05); (44. 56 1. 16) %, (64. 09 0. 81) %, (P < 0. 05), 3 ; (3. 68 0. 07) % (41. 82 0. 56) %, (P < 0. 05), 0. 035 ggml [ ] ; ; ; ; [] S828. 3 + 4 [] A [ ] 167129387 (2006) 0620041205 1956 Po lge [1 ],,,, -, ;, [223 ],, [4 ],,,,,,,, [528 ],, Bayard [9 ],D al2 im ata [10 ] W oelders [11 ] ; E lm an [12 ] E im an [13 ] - ; [14 ] - ; Fernando [15 ], TCG, 3, -,, 1 1. 1 1. 1. 1 1. 5 2 Ξ [ ] 2005210227 [ ] (2004K022G11203, 2005K022G03203) ; (05ZR 100) [ ] (1976- ),,,, [ ] (1956- ),,,,, E2m ail: liqingw angysu. @yahoo. com. cn
42 () 34 8,, 1 1, 0. 7 1. 1. 2: D 2, (F ITC2PNA ) (P I) CTC 3 Sigm a ; ; : B IO TRON 2 ICS ; Barnan t ; ; PH S23C ph ; O SM OM A T 030 ; 1. 1. 3 ( 1) TCG [16 ] 1. 1 g, 1. 48 g, T ris 2. 42 g, 100 ml, ph 6. 21, 286 mo sm ( 2) BT S 3. 7 g, ED TA 10. 125 g, 0. 6 g, N ahco 3 0. 125 g, KC l 0. 007 5 g, 0. 06 g, 0. 1 g, 100 ml, ph 7. 25, 315 mo sm (3) P I( ) 0. 004 g P I 2 ml PBS, 0. 45 Λm, - 20 (4) - 0. 675 6 g, 0. 367 6 g, 50 ml, 150 mo sm ( 5) 123 0. 000 4 g 123 2 ml (DM SO ), 0. 45 Λm, (6)CTC T ris 0. 024 2 g, N ac l 0. 076 g, L 2 0. 006 g, CTC 0. 039 g, 10 ml, ph 7. 8, 0. 45 Λm, (7) (F ITC2 PNA ) 0. 001 g F ITC2PNA, 10 ml PBS, 0. 45 Λm, 1. 2 375 mmo lgl, TCG, 0. 035, 0. 070, 0105, 0140 ggml, TCG 3,, P I2 123 F ITC2PNA CTC 3, (HO ST ) 1. 3, 50 ml, (800 g, 10 m in), BT S, 15 15 3 h, (800 g, 10 m in),, g (TCG + 20% ), 5 2 h, g (3% g ), 5 1 h, (0. 25 ml ), 1 gm in 5-5, 3 cm 15 m in, 1. 4 1. 4. 1, 37, 30 s, 10 ΛL,, 400 ( ) 1. 4. 2 BT S, 3 10 6 gml 100 ΛL H EPESgBSA [16 ] 1. 5 ml, 10 ΛL P I 1 ΛL 123, 10 m in, 50 ΛL, 37 30 m in 10 ΛL,, (400 ) 488 nm,, ( ) ( ) 1. 4. 3 - (HO ST ), 1 10 6 gml, 37 30 m in, 20 ΛL, 400,, 200 1. 4. 4L am ia [17 ], F ITC2PNA, PV P,
6 : 43 800 g 6 m in, ; PBS, 1 10 6 gml, 30 ΛL,, 10 m in, 30 ΛL F ITC2PNA, 37 30 m in, PBS, (10% PBS ),, 1. 4. 5BT S, 1 10 6 gml, 400 ΛL 1 ml 30 ggl PV P, 500 g 6 m in,, (1) 200 ΛL PBS, 200 ΛL CTC, 30 s, 36 ΛL 12. 5%, 10 ΛL,,, 400 (2) 200 ΛL TCM, 3 h, 200, 1. 5 SPSS O ne2w ay ANOVA 2 2. 1 3 1,, 0. 035 ggml, (P < 0. 05) ;, ;, (46. 34 0. 52) % (43. 58 0. 49) %, TCG 1 3 - (n= 21) T able 1 V iability,mo tility and the percentages of m itochondrial activity sperm after frozen2thaw ed w ith disaccharides(n= 21) Group g (g ml - 1 ) Concentration g% V iability g% M o tility M itochondrial activity TCG Contro l 0 31. 61 0. 97 a 31. 26 0. 37 a 31. 62 1. 17 a T rehalo se 0. 035 46. 34 0. 52 b 41. 38 0. 39 b 44. 56 1. 16 b 0. 070 32. 40 1. 01 a 31. 06 0. 52 a 31. 49 0. 96 a 0. 105 21. 63 1. 02 c 12. 03 0. 04 c 21. 08 0. 46 c 0. 140 18. 96 1. 57 d 12. 03 0. 04 c 16. 97 0. 61 d Sucro se 0. 035 43. 58 0. 49 b 41. 29 0. 43 b 41. 08 0. 53 e 0. 070 37. 29 1. 12 e 32. 03 0. 09 a 33. 38 1. 21 a 0. 105 21. 06 0. 38 cd 11. 68 0. 26 c 20. 61 0. 52 c 0. 140 17. 97 0. 62 d 11. 68 0. 26 c 17. 05 0. 78 d L acto se 0. 035 38. 29 0. 37 e 32. 33 0. 45 a 35. 76 0. 61 a 0. 070 27. 39 0. 52 f 26. 89 0. 75 a 31. 48 0. 46 a 0. 105 20. 09 0. 47 cd 11. 00 0. 00 c 19. 81 0. 58 c 0. 140 18. 67 0. 65 d 11. 00 0. 00 c 14. 07 0. 65 d : (P < 0. 05) N o te: V alues w ith different sm all letters w ith in the sam e co lum n differ significantly. Sam e in the fo llow ing tables. 2. 2 3 1, 3 0. 035 ggml,, TCG (P < 0. 05),, (P > 0. 05) (P > 0. 05), 2. 3 3 1, 0. 035 ggml,, ( 44. 56 1. 16) %, (41. 08 0. 53) % (35. 76 0. 61) %, (P < 0. 05),
44 () 34 TCG (P < 0. 05) 2. 4 3 2, 3 0. 035 ggml, Group (P < 0. 05), (43. 51 1. 78) % (43. 37 0. 72) %, ( P < 0. 05) ; 3, 2 3 - (n= 21) T able 2 Effect of 3 disaccharides on the param eters of m em brane integrity, acro som e integrity g (g ml - 1 ) Concentration and capacitation of frozen2thaw ed sperm M em brane integrity A cro som e integrity Rate of capacitation sperm Befo re capacitation A fter capacitation TCG Contro l 0 31. 40 1. 13 a 38. 85 0. 72 ac 8. 72 0. 85 a 29. 23 0. 52 a T rehalo se 0. 035 43. 51 1. 78 b 64. 09 0. 81 b 3. 68 0. 07 b 41. 82 0. 56 b 0. 070 27. 43 3. 59 c 65. 38 1. 16) b 4. 41 0. 16 b 28. 95 0. 66 a 0. 105 19. 23 0. 96 d 66. 02 2. 04 b 4. 29 0. 24 b 17. 69 0. 85 c 0. 140 14. 92 0. 51 de 67. 49 1. 62 b 5. 17 0. 62 b 15. 67 0. 09 c Sucro se 0. 035 43. 37 0. 72 b 38. 91 0. 53 ac 7. 61 0. 83 a 38. 99 0. 57 c 0. 070 33. 29 0. 62 a 39. 27 0. 67 ac 8. 59 0. 63 a 33. 88 0. 69 d 0. 105 19. 31 0. 74 d 40. 63 0. 52 a 10. 65 0. 57 c 17. 35 0. 73 c 0. 140 17. 58 0. 72 d 41. 09 0. 53 a 10. 28 0. 73 c 12. 90 0. 18 e L acto se 0. 035 35. 07 0. 91 a 36. 24 0. 51c 7. 39 0. 61 a 34. 71 0. 42 d 0. 070 30. 56 0. 47 c 36. 48 0. 88 c 10. 97 0. 48 c 25. 84 0. 46 a 0. 105 18. 54 0. 58 d 38. 51 0. 71 ac 11. 56 0. 71 c 16. 83 0. 91 c 0. 140 14. 49 0. 75 e 39. 37 0. 63 ac 12. 29 0. 74 c 14. 79 0. 73 e 2. 5 3 2,, 0. 035 ggml, (64. 09 0. 81) %, (P < 0. 05) ;,, (P > 0. 05) 2. 6 3 2,,, - (P < 0. 05) ;, 3 (P < 0. 05), 0. 035 ggml,, (41. 82 0. 56) %, (38. 99 0. 57) % (34. 71 0. 42) % 3, 0. 035 ggml,,,, [15, 17218 ],,,, E lm an [12 ],, 0. 035 ggml,,, ;, E lm an [12 ],,,,,, [15 ],,
6 : 45,,, Garcia [19 ],, -,,,,,, Jelinkova [20 ],,,, [ ] [ 1 ] Po lge C. A rtifical insem ination in p ig[j ]. V et Pec, 1956, 68: 62276. [ 2 ] L isa M T hurston,w illiam V Ho lt, Paul F W astson. Po st2thaw functional status of boar sperm atozoa cryop reserved using th ree contro lled rate freezers: a comparison[j ]. Theriogeno logy, 2003, 60: 1012113. [ 3 ] W oelders H, M atth ijs A, Zuidberg C A, et al. C ryop reservation of boar sem en: equilibrium freezing in the cryom icro scope and in straw s[j ]. Theriogeno logy, 2005, 63: 3832395. [ 4 ] Kum ar,m illar,w aston. T he effect of coo ling rate on the survival of cryop reserved bull, ram, and boar sperm atoaoa: a comparison of two contro lled2rate coo ling m achines[j ]. C ryobio logy, 2003, 46: 2462253. [ 5 ],. [J ]., 2003, 5 (1) : 78280. [ 6 ],. [J ]., 2001, 21 (3) : 2062209. [ 7 ] A lex, H ans. P reservation m echanism s of trehalo se in food and bio system s[j ]. Co llo ids and Surfaces B: B io interfaces, 2002, 57: 169521706. [ 8 ],. [J ]. :, 2004, 19 (6) : 63266. [ 9 ] Bayarad, E sther, Kath leen. Comparison of glycero l, o ther po lyo ls, trehalo se, and raffino se to p rovide a defined cryop ro tectant m edium fo r mouse sperm cryop reservation[j ]. C ryobio logy, 1998, 37: 46258. [ 10 ] D alim ata, Graham. C ryop reservation on rabbit sperm atozoa using acetam ide in com bination w ith trehalo se and m ethyl cellulo se[j ]. T he2 riogeno logy, 1997, 48: 8312841. [ 11 ] W oelders,m atth ijs, Engel. Effects of trehalo se and sucro se, o smo lality of the freezing m edium, and coo ling rate on viability and intact2 ness of bull sperm after freezing and thaw ing[j ]. C ryobio logy, 1997, 35: 932105. [ 12 ] E lm an, A boagla, T erada. T rehalo se2enhanced fluidity of the goat sperm m em ebrane and its p ro tection during freezing[j ]. B io logy of Re2 p roduction, 2003, 69: 124521250. [ 13 ] E im an, T akato. Effects of the supp lem entation of trehalo se extender containing egg yo lk w ith sodium dodecyl sulfate on the freezability of goat sperm atozoa[j ]. Theriogeno logy, 2004, 62: 8092818. [ 14 ],,,. [J ]., 1997, 28 (2) : 1202125. [ 15 ] Fernando, M argareta, Szabo lcs, et al. D eep freezing of concentrated boar sperm fo r intra2uterine insem ination: effects on sperm viability[j ]. Theriogeno logy, 2005, 63: 132021333. [ 16 ] E rik sson, Rodriguesz2M artinez. Effect of freezing and thaw ing rates on the po st2thaw viability of boar sperm atozoa frozen in F lat Pack s and M axi2straw s[j ]. A nim al Rep reduction Science, 2000, 63: 2052220. [ 17 ] L am ia, D aniel, L aetitia. Bull sem en in v itro fertility after cryop reservation using egg yo lk LDL : a comparison w ith Op tidyl, a comm ercial egg yo lk extender[j ]. Theriogeno logy, 2004, 61: 8952907. [ 18 ] F riedler S, Giudice L C, L am b E J. C ryop reservation of em bryo s and ova[j ]. Fertil Steril, 1988, 49: 7432764. [ 19 ] Garcia D e Castro A, T unnacliffe A. Intracellular trehalo se imp roves o smo2to lerance but no t desiccatio to lerancein m amm alian cells[j ]. FEBS L etters, 2000, 487: 1992202. [ 20 ] Jelinkova L, Selm an H A, A rav A, et al. Tw in p regnancy after vitrification of 22p ronuclei hum an em bryo s[j ]. Fertil Steril, 2002, 76 (2) : 4122414. ( 51 )
6 : PA 51 C lon ing and imm unop ro tection of PA recom b inan t p ro tein of B acillus an th racis X IE Y ing-guo 1, 2,W U X iu-fang 1, 2,W U Su-q in 2,W ANG X i- liang 2, HE W e i-m ing 1 (1 Colleg e of A nim al S cience and T echonology,n orthw est A & F U niversity, Y ang ling, S haanx i 712100, Ch ina; 2 D ep arm ent of Imm unology of R eseach Institute of M icrobiology and Ep idem iology A cademy of M ilitary M ed ical S cience, S tate K ey L aboratory of P athogen and B iosecurity,b eijing 100071, China) Abstract: PA gene amp lified w as from B acillus an th racis by PCR and then cloned in to pet 28a (+ ) vecto r. T he recom b inan t p lasm id pet 28a (+ ) 2PA w as con structed and exp ressed and the imm unogen icity and imm unop ro tection w ere studied. T he resu lts show that the length of PA gene is abou t 2 205 bp and rel2 ative mo lecu lar m ass of the exp ressed p ro tein is 83 ku; the rpa has good imm unogen icity, and specificity of an tibody cou ld be detected after 10 d imm un ization, and the an tibody levels rise m arkedly, 3rd to 1 12 800; the rpa p ro tein stim u lates the specificity of an tibody b inding to mou se IgG12based; the rpa p ro tein can p romo te the grow th and m u ltip lication of the mou se sp leen cells in v itro. T he rpa imm un ization group can sign ifican tly increase CD + 4 mou se sp leen cells and the imm une respon ses tend to T h22type respon se, show ing that the rpa mo st m ediate to humo ral imm un ity. T he rpa is fairly imm une p ro tective, the p ro tec2 tion rate being 50%. Key words: B acillus an th racis; p ro tective an tigen; imm un ity; genetic engineering vaccine ( 45 ) Abstract ID: 1671-9387 (2006) 06-0041-EA Effect of trehalo se, sucro se and lacto seon on cryop reservation of boar sperm ZHANG Shu- shan 1,L I Qing-wang 1, 2,L I Gang 1, CHEN X iao-yu 1, HU J ian-hong 1, HAN Zeng- sheng 1,W ANG L i-q iang 1 (1 Colleg e of A nim al S cience and T echnology, N orthw est A & F U niversity, Y ang ling, S haanx i 712100, Ch ina; 2 D ep artm ent of B iolog ical Eng ineering, Collage of Env ironm ent & Chem ical Eng ineering, Yanshan U niversity, Q inghuangdao, H ebei 066004, China) Abstract: In th is study, the effects of adding th ree disaccharides (trehalo se, sucro se and lacto se) in the cryop reservative so lu tion w ere compared. T he resu lts w ere summ arized as fo llow s: T he effect of boar se2 m en cryop reservation w as m arkedly imp roved w ith th ree disaccharides, and their op tim um level w as 0. 035 ggml ; A t the level of 0. 035 ggml, the viab ility, mo tility, the percen tages of m itochondrial activity and m em b rane in tegrity of frozen2thaw ed sperm w ith trehalo se w ere (46. 34 0. 52) %, (41. 38 0. 39) %, (44. 56 1. 16) % and (43. 51 1. 78) % respectively, and there w as no sign ifican t difference betw een tre2 halo se and sucro se (P > 0. 05), and bo th of them w ere h igher than that of lacto se group and the con tro l (P < 0. 05) ; T he percen tage of in tact acro som e of trehalo se group w as (64. 09 0. 81) %, and h igher than that of the sucro se, lacto se group, and the con tro l (P < 0. 05), and increased w ith the increasing level of th ree disaccharides; T he percen tages of capacitating boar sperm befo re and after capacitation treatm en t w ere (3. 68 0. 07) % and (41. 82 0. 56) %, and w as better than that of lacto se group and the con tro l(p < 0. 05). To sum up, the effect of imp roving the frozen2thaw ed sperm quality w ith trehalo se w as superio r to sucro se, lacto se and TCG con tro l, and the op tim um level w as 0. 035 ggml. Key words: boar sem en; cryop reservation; trehalo se; sucro se; lacto se