HOI NGH! KHOA HOC CONG NGHE SINH HQC TOA ' > -
logi B: cd h> 2-3 Idp td bao cdn noan bao quanh frimg, cdc t l bdo ndy lidn kit khdng ch$t chS. cd noi bj mit mOt phin t l bdo nguydn sinh chit dong diu nhung hoi tdi mdu d vilng ngogi vi tn>ng. todn bd frung nhin ft frong vd ft flly a$n ha^
Chi chpn nhdng frdng logi A. B d l nudi. Mdi frudng nutt Sidnh thyc trtmg Id 199 hepeses b l sung 10% FBS (fetal bovine senjm) vdi 40-50 frdng /Igidng 500 p'- Tning lyn sau khi nudi 44-46 gid frong nidi tnrdng 199 10% FBS cd thi dgt din giai dogn ttidnh ttiyc (ddnh gid bing sy xuit hidn ttie cyc ttid nhit). Nhung frdng thdnh ttiyc vd cd td bdo chit ddng nhit duyc lya chpn d l ttiy ttnh 6ng nghi$m.
Thu t l bdo mdng Idt dng dan trdng
T l bdo mdng Idt dng din frdng (Hinh 1-a) duyc ttiu tir 6ng din frdng cila lyn rfiua ttidnh ttiyc. 6ng din frdng ttiu Kr B md. diryc vdn chuyen vd phdng ttil nghidm frong 1-2 gid. Sau dd duyc n>a 3-4 lln bong ni/dc mudi sinh \^ bd sung khang sinh gentamycin vdi ham luyng 240 mg/l vd n>a Igi 2-3 lan frong mdi frudng PBS d 37°C. Dung kdo vd kgp vd triing cat pt^n md vd td didc lidn k i t quanh dng din trdng. DCing kdo vd frOng d t hai diu 6ng din frung (chpn phin dng din frdng phia dirin), ttiu liy t l bdo mang Idt dng din frdng vd dem nudi trong dia 4 giing c6 chda mdi frudng 199 Na. 500 pi / gieng. Thu lay nhung t l bdo c6 khd nang di ddng vdo ngdy hdm sau. Thay mdi frudng sau 1- 2 ngdy.
Thu t l bdo syi cila bdo fiiai c h u ^
Phuyng phdp thu t l bao syi cOa bdo ttiai chudt duyc ttiyc hign ttieo phuong phap ciia Robertson (1987). Qui frinh duyc md td mdt each ngin gpn nhu sau: Phdn tdch ttiai td Kr cung, tdch ndng tdng ^ a i vd chuyin sang dTa pefri chda PBS vd frimg. Diing kgp bd diu, chdn, nji tgng. DOng dao ho§c kdo vd trCing cat de tgo cdc milng md cd kich ttiudc 1 mm xi mm. Thdm 5 ml ttypsin /EDTA 0,25% m i l 10 ttiai. DOng xilanh togi 10 ml vdi kim cd 18. 23 hOt Idn hOt xudng.
Thdm luyng DMEM bing luyng trypsin d l trung hda trypsin.
Nudi td bdo syi cGa bdo ttiai chudt (Hinh 1-c) trong dta petri NunC (Dan Mgch) dirdng kinh 6 cm hole 9 cm.. Ciy ehuyin td bdo sau 2-3 ngdy liic mdi nudi, sau dd ed 3 ngdy ciy chuydn mdt l l n . Td bao ttiudng duyc ddng Ignh d nhung lin ciy chuyin ttilp d l dung khi cin. Ddi vdi td bao syi cOa bdo tiiai chudt, tnrirc khi nudi kit hyp phdi xd [^
bing mytomycln dd dc chd sy phdn chia td bdo. M$t dd td bdo trong dia 4 gidng Id 300.000 - 400.000 t l bdo vd frong dfa trdn kich ttiudc 6 cm Id 600.000 t l bdo la cd thd dung ldm td bdo d$m cho nudi kit hyp vdt frdng vd phdi.
Thv tinh dng nghidm (IVF)
Mdi tnrdng ttiy tinh Id mdi frirdng Plg-FM cdi bidn cd bd sung 2 mM caffein khan vd 5 mg/ml bovine semm albumin (BSA; Fractfon V, Sigma). Ky thu§t IVF duyc Idm theo phuong phdp ciia Kikuchi vd cdng sy (2002). Kjr thu|t ndy duyc md td mdt cdch ngin gpn nhu sau: Tnrng sau khi nudi frong mdi frudng 199 10% FBS 44 - 46 gid, td hyp frdng - t l bdo cdn nodn (COCs) duyc chuyin vdo gipt 100 pi mdi trudng thy tinh FM, gipt thy ttnh duyc ngp trong dia trdn vd duyc phu diu paraffin dm (Paraffin Liquid, light; Nacalai Tesque. INC., Kyoto, Japan). Mdi gipt thy tinh ehda 10-15 tn>ng duyc thy tinh bing tinh tning ddng Ignh - gidi ddng cOa lyn dye gidng Landraee. Tinh trung duyc u trong hJ 37°C phU diu Im 15 philt. Ndng dd tinh cudi cOng trong gipt thy tinh Id Id ICr tinh frOng/ml. Trdng duoc Ci vdi tinh tning trong 2,5-3 gid d 38.5'C, 5% COz. Ngdy ttiy linh duyc tinh Id ngdy 0.
Nudi phdi In vitro (IVC)
Sau khi trdng u vdi tinh triing 2,5-3 gid, cdc t l bdo t l bdo e$n noan vd tinh trung bdm quanh frdng duyc tdch khdi t l bdo trdng bang cdch dOng kim thdy tinh ed kfch thudc phu hyp nhg nhdng htlt lln. ttili xudng. Trdng sau khi tdch sgch td bdo cdn noan duyc chuyin vao mdi frudng IVC: Mdi trudng IVC gdc Id NCSU-37 cdi ttdn bing cdch ^dm 0.4% BSA (theo Uil tich) vd 50 pM p-mercaptoethanol. Cd hai logi mdi frudng IVC: NC-PytLac (MSi tnjdng IVC ca bdn cd bd sung 0,17 mM natri pyruvat vd 2.73 mM natri lactac) vd IVC-glu (mdi ttudng co bin cd bd sung 5,55 mM glucose). Phdi duyc nudi trong td nufli 38,5''C. 5% CO2 trong mdi tnrdng IVC PyrLac td ngdy 0 d i n ngdy 2, sau dd duyc chuyen sang nudi Irong mdi trudng cd bo sung glucose cho den ngdy 6.
Bd tri thi nghi|m
Thi nghidm 1: Anh hudng cOa kich thudc nang tdi sy phat triin phfli lyn sau ttiy tinh dng nghidm. TnJng lyn duyc thu tij hai nhdm nang cd ki<^ thudc 2-3 mm vd >3 mm, nudi thdnh thyc theo nhdm vd ttig tinh bing tinh tiling ddng Ignh - giai ddng. Sau ttiy tinh. irtrng duyc nudi trong mfli frudng nudi cd bd sung t l bao syi cOa bdo thai chudt.
Thi nghidm 2: Anh hudng cua td bdo d$m tdi sy phdt triin phdi lyn sau thy tinh dng nghi§m (ttif nghi|m vdi nang cd kich thudc >3 mm). Trdng lyn ttiu td nang kich thudc >3 mm duyc nudi thdnh thyc, chpn nhdng tiling dii tiiu chuin thv tinh Ing nghidm. Trdng lyn sau ttiy tinh duyc chia Idm 3 nhdm nudi
Nhdm 1: Mfll tnrdng nudi phdi khflng ed te bdo d$m (ddi chdng).
Nhdm 2: Mdi tmdng nudi phfli cd t l bdo mdng Idt dng din tning (Hinh 1-b) Nhdm 3: Mfli trudng nudi phfli cd td bdo syi cCia bdo thai chuflt
Cdc chi tidu ttieo ddi Id ty Id chia, t# Ig phfli 2-16 t l bdo, ty Ig phfli ddu. ty Id phdi nang it ngdy thd 6 sau thy tinh so tning chia
Tf 1$ chia {%) =
Ty Id phfli 2-16 t l bdo (%) =
tong trdng thf nghidm sd phfli 2-16 t l bdo
-QC CONG NGHE SINH HOC TOAN QUOC 2013
sd trdng chia sd F^fli dau TJIg phfli ddu {%) =
Ty Id phfli nang (%) =
sd t n ^ g diia so phfli nang so trdng chia Xi>l:^s6li$u
sd ligu duyc xd ly bing phin mem excel, thing kd mfl ta va phan h'ch phuong sai mflt nhdn td {Anova single factor) K ^ QUA v A T H A O L U A N
Phfli d ngdy ttiu 6 sau ttiy tinh (Hinh 1-d, e, f) duyc ddnh gid cdc ehi tieu ttieo ddi. TJ le chia, t j Id phfli 2-16 t l bao vaty 1$ phfli dau khdng c6 sy khac bi$t giua t n ^ g thu td nhdm nang 2-3 mm vd nhdm nang >3 mm. Tuy nhidn ty Id phdi nang d nhdm nang >3 mm cao hon cfly nghTa so vdi nhdm nang 2-3 mm tuong dng Id (27,05 ±2.16; 9.95 ±1,63) (Bang 1).
Bdng 1: Anh hudng cCia kich thudc nang tdi sy phdt trien phdi lyn In vitro
Kfch thudc nang Ting frdng T^ 1^ chia (%) TJ 1$ ph6i 2-16 te bdo (%) Tf 1$ Ph6i ddu (%) Ty 1$ phdi nang (%) Nang 2-3 mm 145 61.71 ± 3.76 38.5± 3,77 34,45± 3.49 9.95" ± 1.63 Nang >3 mm 413 69,74 ± 3.38 37.93 ±4.61 31,25±4.23 27,05''± 2.16 Cdc chd cd; a, b theo cgt thi hi$n si/ khic bi$t cd y nghTa vtrip<0,05
Sy thdnh thyc cda tiling dflng vdt cd vO cd liln quan tdi sy phdt triin nang budng trdng. Mdi lien hg ndy da duyc xdc nhdn trong didu kidn In vitro it rit nhieu lodi bing each nghidn cdu dnh hudng eOa kfch ttiudc nang tdi kha nang phdt tiiln phdi cOa trdng sau khi nudi thdnh ttiyc in vitro, ttiu tinh vd nudi phdi. Dilu ndy xdy ra vdl trdu bd. dd. cdu. Tning thu td cdc nang eOa frdu bd cd kfch thudc td 4-6 mm hogc > 6mm, cda dd cd kfch thudc >5 mm. ciia cdu cd kfch ttiude >3- 5 mm cd khd ndng phdt triin din giai dogn phdi hon Id frdng thu td nhdng nang cd kich thudc nhd (frau bd 1-3 mm, dd 2-3 mm, cdu 1-2 mm). Trtrng eCa dflng vdt c6 vii frdi qua sy thay doi ed y nghTa bdn frong nang trimg d$c bidt sau khi ttiilt ldp b'nh byi va ddn thdi diim njng tiling (Khatir et al..2007}. Doi vdi Igc dd, t j 1$ tning phdn chia d nhdm nang to (>6 mm) Id 72%. cao hon so vdi nhdm nang nhd (3-6 mm) Id 59%, P < 0.05. TJ 1$ frdng ttiy tinh phdt friln din giai doan phdi nang tuong duong cua hai nhdm Id 35% vd 20% (P < 0.05). Khi ciy nhung phdi nang cda cdc nhdm ndy vdo con nhdn cd ddng dye ddng pha, kdt qui cho thiy khdng cd phdi nang nao cua nhdm nang 3-6 mm phdt triin 6dp. trong khi 68% phfli eOa nhdm nang >6 mm phdt triin din giai dogn mang thai 25 ngdy, 53% tnjng sd dd phdt triin ddn giai dogn mang thai 2-4 thdng, 5-7 thdng va 47% mang thai vd sinh ra con binh thudng. khde mgnh (Khatir et al.. 2007).
Nghidn cuu cua Yun QIAN vd ddng tdc gid (2001) cung chi ra ring: kha ndng phat tiidn cda trdng lyn sau khi Uiy tinh Ing nghidm phy thuflc vao kfch thudc nang. TJ le chia cao hon vd sy can xdng hon cda cda phfli 3-4 t l bao diryc quan sdt d nhflm nang ed dudng kinh £4 mm so vdi nhflm nang 2-2.9 mm. Mac dil sy can xdng cua cac phdi 6-8 td bdo vd 12-16 t l bdo gida cdc nhdm nang khdng cd sy khdc bidt, phfli d nhom nang 2-2.9 mm bj ddng Igi d giai dogn 8 td bdo;
phdi d nhdm nang 3-3 9 mm bj ddng Igi d giai doan 16 td bdo vd khdng cd phdi ndo phdt tnIn din giai dogn phdi ddu.
Cdc kit qud nghidn cdu cda chung tfli khang dinh thdm vai fro cua kfch thudc nang trdng ddi vdi sy phdt trien tiep theo ciia frdng lyn sau ttiy tinh dng nghidm.
Kit qud nufli phdi vdi cac logi t l bdo dgm cho ttily khdng co sy khdc nhau vd t j Id chia vd t j 1$ piidi ddu gida 3 nhdm:
nhdm ddi chdng (nhdm 1). nudi kit hyp vdi t l bdo mdng lot dng dan trdng (nhflm 2) hay t l bdo syi cua bao ttiai chuflt (nhdm 3). Kit qud h/ong dng lln luyt Id 63,79 ± 3.49. 66.88 ± 4,15 vd 63.48 ± 3,19; 30.29 ± 4.49; 40,61 ± 3.88; vd 38,89 ± 2.31 (P > 0.05). Nhdm 2 vd 3 cd t j Id phfli 2-16 t l bdo, ty Id phdi nang tuong duong (25,92 ± 4.04. 33,85 ± 2.85 so vdi 24,49 ± 1,61; 27,74 ± 1.83. P > 0.05). Tuy nhien ty Ig phfli 2-16 td bdo cua nhdm 1 Igi cao hon cd J nghTa so vdi nhdm 2 va 3 (53.49 ± 6,78) ti^ng khi t j Id phdi nang lai ttilp hon nhom 2 vd 3 (16.21 ± 2.75) vdi P < 0.05. Didu ndy cd thi gidi Uifch Id t l bdo ddm giup phfli d nhflm 2 va 3 vuyt qua duyc giai dogn 2-16 t l bdo va thuc diy chiing phdt friln din giai dogn phdi nang.
Bdng 2: Anh hudng cua t l bdo dfm tdi sy phit triin phoi lyn sau thu tinh dng nghidm
Logi t l bdo d^m Tdng tn>ng T91$ chia (%) TJ 1$ phK 2-16 t l Ty l# PhSi ddu (%) Ty le phfli nang (%) t>do(%)
Odi chdng 712 63,79± 3.49 53,49' ± 6.78 30.29 ± 4,49 16.21 " i 2.75 T l bdo mdng Idt 615 66,88 ± 4,15 25,92° ±4.04 40.61 ± 3,88 24,49''± 1,61 I n g dfin trdng
T i b d o s y i c C i a 1352 63,48±3,19 33,85''±2,85 38,89±2.31 27,74''±1,83 bdo thai chuOt
Cic chO" cii a. b trong cOng cgt thi hi^n su khic bi$t co y nghia vdiP<0,05
HUI NLiM! t \ n u A n y u UUNU N l i M t ail^H HOC T C
Kit qud nghidn cdu cua chiing tdi phu hyp vdi kit qud eua LTflrc vd ddng tdc g'ld (2008). fi Id tning ttiy tinh vd phdn chia thdnh 2 t l bdo d nhdm (Landraee x Landraee) dat 75%. cdn t j Id phfli phdt hidn tfli giai dogn phfli nang Id 20% . Theo White KL vd ddng tdc gid (1989), phfli duyc nudi trtwig mdi budng c6 b l sung PEF(pordne fetelfibroblast monolayer), POEC (porcine oviductal epittieliai cells), PEF-POEC c6 27%, 70% vd 67% ttieo ttid ty phdt triin din giai dogn phdi nang 38%. 11 %, 7% vd 77% phS 2 t l bdo va 4 t l tiao khdng phdt frien tiip sau Idii phdn chia 1 -2 lln khi nudi ttong mdi frudng PEF. POEC. PEF-POEC vd DMEM (Dulbecco's Modified Eagle Medium) ttieo ttid ty. Ket qud ridy duyc giai ttiieh ttieo Gandoffi va Moor (1987) Id cd thd nhdng t l bdo ndy hd fry sy phdt friln binh ttiudng trong nudi in vitro. Cung theo Polge (1982) ttil sy ^ d t triin phdi in vitm ir lyn ed ngudng la giai dogn 4 t l bdo. T l bao d§m mdng Iflt dng din frdng cung cip cdc ylu td can thiet cho sy phdn chia cda [rfidi lyn vuyt qua giai doan 4 t l bdo vd sy phdt triin tiip ttieo tdi giai dogn phfli nang.
Hinh 1. T l bdo d$m vd sy phdt tiiln phdi lyn TTON. a-Tl bdo mdng I6t dng din frdng (ODT). b-.NuAi phfii kit hyp vdi t l bdo ODT.
c-Tl bdo spi ciia bao thai chu$l d- Phdt giai dogn 2-4 t l bdo. e- Phfii ddu. f-Phoi nang
K ^ L U A N
Kfch thudc nang budng trdng lyn cd dnh hudng tdi sy phdt tridn tidp theo cua tning lyn sau thy tinh dng nghigm: Tning ttiu hi nhung nang cd kleh thudc >3 mm cho ty Ig plidi nang cao han so vdi trdng thu tii nang ed kfch thudc 2-3 mm.
Chiing tdi de xuit ndn chpn bdng thu td nhung nang cd kfch thudc >3 mm diing trong vigc tgo cdc sdn phim dng nghidm.
T I bdo dgm Idm tang tylg phdi phdt bien vuyt qua duyc giai dogn 2-16 td bdo vd choty Id phdi nang cao hon so vdi dii;
chdng. Tuy nhidn t j Id ndy khdng cd sy khdc bigt khi dung cac logi te bdo dgm tren. Do dd, chung toi d l xuit ndn bl sung don Id ho^e cd hai logi t l bdo nay frong nufli kit hop vdi phdi lyn in vitro dd tang ty Id va chit luyng phdi phdt triin ddn giai dogn phfli nang.
; sif ldi Irfr kinh phi aia Qu 'phdi Inen Khoa hoc CO gid In y sinh"
a Cong ngh? quoc gia cap cho di Idt "N^iin Liti c i m an: Cong trinh ndy duffc hodn ihdnh vi
cau plt^ triin cong nghi cdy nhdn vd nhdn bdn v6 TAI Ligu THAM K H A O
Annadie KIdson, Eric Schoevers. Pieter Langendijk, Jos Verheijden, Ben Cdenbrander, Mart Bevers (2003). The effect of oviductal epithelial cell co-culture during in vrtro maturation on sow oocyte morphology, fertilizaton and embryo development. Theriogenotog/
(59): 1889-1903.
Elizat>eth J Robertson (1987) Teratocarcinomas and embryonic stem cells: a practical approach. Oxford University Press. USA.
Khatir H, Anouassi A, Tibary A (2007). Effect of follicular size on in vifro developmental competence of oocytes and viability of embryos afte transfer in the dromedary (Camelus dramedarius) Anim. Reprod Sci (99): 413-420.
Kikuchi K, Onishi \ Kashiwazaki N, Iwamoto M, Noguchi J, Kaneko H, Akita T. Nagai T (2002). 'Successhit pjglet production after transfer of blastocysts produced by a modified in vitro system" Biol Reprod 66(4): 1033-41.
Leibfned L and First NL (1979) Characterization of bovine follocuiar oocytes and their at»lity invitro. J Anim Sci (48). 76-86.
Lloyd RE, Romar R. Matds C, Guti6rrBZ-Adan A, Holt WV, Coy P (2009), Effects of oviductal fluid on the development, quality, and gene expression of porcine blastocysts produced in vitro. Reprod 137(4):679-37
I ,.', ,., -1. 1 - - • . - - , i ^ v j i i o i N V j n ^ o i M n n v ^ . - I W A N u u w i j ^ u i s j
•*^slia-Ksiqa«ew»cz L (2006). Pig embryo production by in vrtro mafairalion and fertilization of ovanan oocytes. A review. J Anim Sa
MalmoutJ Heidari, Atoasali Karimpour Malekshah. Kazem Parivar. Ramezan Khanbabaei. Alireza Rafiei (2011). Effect of Fibroblast Co- cJtire on In vitro Maturatkxi and FertilizaUon of Mouse Preantral Fdlides- J Fettil Stent (5): 1-8.
Polge C (1982). Embryo transplantatkm and preservatron. In: D.JA. Cole and G. R. Foxcroft (Ed) Control of Pig Reproducton. Pp 277-291. Butterworth Scientific, London.
White KL, Hehnke K, Rickonis LF, Southern LL, Thompson DL, JR. and Wood TC (1989). Eariy embryonic Development in vifro by CocuHure vritti owductal ^xthelial cells in pig. Biol of Reprod (41): 425-430.
Yun Qian, Wei Qun Shi. Jia Tong Ding, Bi Qin Fan And Yulaka Fukui (2001) Effect of Follicle Size on Cumulus-Expansion, tn vitro Fertilization and Devetopment of Porcine FoUicglar Oocytes. J Reprod Dev (47): 145-152
Nguyen Th] U d c , Nguyin Viet Linh, Nguyin Vdn Hanh, Qudn Xudn Hdu, Ddng Nguyin Quang Thdnh, Trdn Thi Therm, Nguyin Thi M i n , Biii Linh Chi, Nguyin Tmng Thdnh. Duong DInh Long. Nguyin Khac Tich, Phan Ngpc Minh & Bui Xudn Nguyen (2008). Nghign ctni sdn x u i t phoi lyn mini n$i dja bdng td hyp cdng ngh$ I n g nghidm vd nhan bdn vo tinh. CNSH s6 chuyen san, 6 (4A). 625.635.
EFFECT OF FOLLICLE SIZE AND CELL FEEDER CO-CULTURE ON DEVELOPMENTAL COMPETENCE OF IN VITRO FERTILIZATION PORCINE EMBRYOS
Nguyen T h i H i e p , N g u y e n T h i U o c , N g u y e n Viet L i n h , N g u y e n V a n H a n h , Nguyen T h i H o n g , Nguyen Thi Nhung, Nguyen V a n L a m a n d B u i X u a n N g u y e n *
Institute of Btotechnology. Vietnamese Academy of Science and Technology
SUMMARY
Production of porcine embryos by reproductive biotechnology including in viiro fertiUzation has been earned out at VAST for years.
Inlhispaper, we evaluated the influence of foUicle size and feeder cells on the competence development of tn vitro fertilized porcine oocytes. In the experiment l.porcineoocytes were collected separately from 2-3 mm and >3 mm diameter follicle for maturation and ferbhzation and embryos were cultured in the medium with mouse embryonic fibroblasts. In the experiment 2, the oocytes were collected from >3 mm diameter follicle, matured and fertilized as descnbed in experiment 1. The embryos obtained from experiment 2 were cultured under different conditions: without feeder cells (group 1/control), with mouse embiyonic fibroblast (group 2) and with oviductal epidemial cells (group 3). The result showed that the oocytes from >3 mm diameter fbtlicle and in vitro culture medium with fe»ler cells (group 2 and group 3) increased signlfjcanlly the percentage of embryo devetopment through 2-16 cell and blastocyst stage.
Keywords: porcine embryo, in vitro fertilization (IVF), in vitro culture (IVC) follicle size, cell feeder, blastocyst 'Auffiorh)rcon^spondence: [email protected]
