Tgp chi Cong nghe Sinh hpc 10(2)-24\-245, 20\2
ANH HUdNG CUA NONG BQ HORMONE KICH THICH BAO NOAN VA NONG Dp KHI OXYGEN BEN SU THANH TH^JC TRONG ONG NGHIEM CUA TRUNG DE
Nguyln H u u Dire, Tr^n Thi Binh Nguyen Tnrang Dgi hpc Nong nghiep Ha Npi
TOM T A T
Nghien cuu dirge tien hanh nham ddnh gii anh h u ^ g cua nong dg hocmon kich Ihich bao noiSn (Follicular Stimulating Honnone) v^ Oxy den li IC' thanh thyc cua Uirng de nuoi trong ong nghi?m, Thi nghi?m 1, Trirng de dirge thu tir buong tn'mg ya nu6i 32 h Irong moi truong TCM-199 (Earlc's salt wiih L-Glulamine and NaHC03, Sigma, USA) co b6 sung LH (5 pg/ml), E.sliadiol (1 pg/ml), Gcntamycin (50 pg/ml), huyet thanh bao thai be (10% v/v), Natri Pyruvate (0,25mM) vi FSH v6i cac nong dp khiic nhau (0,05; 0,1;
0,2 pg/mi), 5%C02, 39''C Thi nghi$m 2:Trihigde nuoi 32 h trong moi tru6ng TCM-199 co bo sung cac chit nhu Uong thi nghiem 1 v6\ ndng dg FSH la 0,1 pg/ml va trong hai lii nuoi khic nhau, tii nuoi I (5%CO2+20%O2, 39''C, dg km bao hoa) va lii nuoi 2 (5%C02+5%02, 39°C, dg Sm bao hoa), Ket qua cho th3y, ti l?tnjngdethanhth\ic trong moi truang TCM-199 CO bo sung FSH crn6ngdg0,l va 0,2 jig/ml (54,98 + 9,57 % va 58,57 + 4,59 %) li cao hon (P<0,05) so vdi moi truong c6 FSH d n6ng dg 0,05 ng/ml (39,53 + 9,13%); ti le trimg de thanh thyc trong moi tniong TCM-199 c6 bo sung FSH o n6ng do 0,1 pg/ml, 5 ' ' M C 0 2 + 5 % 0 2 , 39<'C (77,92 + 13,67 %) la cao hon (P<0,05) so voi nuoi trong moi tniong TCM-199 co bo SI ng FSH a ndngdg 0,1 pg/ml, 5%CO2+20%O2, 39''C (59.61 + 5,84 %).
Tir khoa: FSH. nong do. Oxy. trung de, thanh thi/ic trong ong ; nghiem M O D A U
Cong nghe sinh hoc sinh san duoc nghien cim va ling dung phuc vu cho cac muc dich cai tao giong, ho trg sinh san a vat nuoi (Lunovic, 2000; Galli ei ai, 2003) va cho ca cac nghien cihi ve te bao goc 6 nguai (Liu et al, 2009).
Cac cong nghe trong linh virc nay bao gom gay rung tning nhieu, thu phoi, cay phoi, dong lanh phoi, tao phoi trong ong nghiem, cat phoi, nhan dong phoi,...trong do cong nghe tao phoi ong nghiem (in vitro embryo production) diroc xem la cong nghe chia khoa cho cac nghien cmi cuu co ban va img dung a trinh d6 cao hon nhu nhan ban va chuygn gen,
Tai Viet Nam, chua co cong trinh nao nghien cihi ve van de noi tren a doi tugng con de. De co the dJit nSn mong cho cong nghe tao phoi in vilro, chiing toi tap tnmg nghien cihi sir phat Irien in vitro ciia tning de thu tir bu6ng tning.
Hai yeu to co anh huong Ion den qua trinh phat trien in vitro cua trirng de la nong dp hocmon FSH (Follicular Stimulating Hormone) va nong do oxy Irong til nuoi (incubator) dugc chiing toi quan tam va danh gia cu t h i miic d6 tac dgng cua chiing den su
thanh thuc trong ong nghiem ciia trimg de. Qua do, gop phan xay dung quy trinh nuoi thanh thuc trung de v6i hieu qua cao trong dieu kien cua Viet Nam, VAT LIEU VA PHUONG PHAP
Cac hoa chat va moi truong sir dung trong cac nghien cihi duoi day deu do hang Sigma, Gibco cung cap,
Thu buong trirng
Mau buong trimg de Co dugc thu thap trong thang 3-4, buong trimg dugc cat ngay khi con vat vira m6i chet, Mau buong trimg dugc nia lai 03 lan bang nuoc muoi smh ly NaCl 0,9% km (37°C) co b6 sung khang sinh Penicilin (10000 UI/lit).
Streptomycin (0,05 gr/lit). Sau do, chiing dugc bao quan a nhi^t dg 37°C va van chuyin vk phong thi nghiem trong vong 2-4 h.
Khai t h i c trfrng tir buong triing
Bu6ng tning dugc riia lai bang dung djch PBS (Phosphate Buffered Saline) 4m (37''C) c6 thanh phan (diing de pha 01 lit) g6m NaCl (8,0 gr), KCl (0,2 gr), Na2HP04 (1,15 gr), KH^PO^ (0,2 gr).
Glucose (1,0 gr), CaCl2.2H20 (0,132 gr), MgCl2.7H20 (0,121 gr), Natri Pyruvate (0,036 gr).
241
Penicilin (10000 Ul/lil). Slreplomyein (0,0'^ gr/IK), pH 7.3,
Trimg Irong eac naim ilii-.K- llui King each efll liVp bui'ing tiimg (dimg dan nut tai ih.inli LIIC din'ii^; Ming song iicii kluip be 11^11 buont; iiiiiij!) Khi LTU. buong trimg dirge ng;im ining inoi irmnig r C - l 9 9 Hepes (SiLini.i) IIUIIL: dugc jzi.u phnrig khoi buong trirng dirge MH tim tren kliih hicn vi sm nin Nikon SM/lti(iii
(,)ii,i trinh soi iim imng tren kinh hicn vi dirge uen hanh lioiig buimg i.i\ \ o IniO!: 1 ;iluoiico | M \ )
\ a tren kinh hien \i luiin luon cii biiii am 17'C 1 ok.u Hit (Nhiit Ban), t i n nliihig iiinig co LIKII lugng loi (l(T]i le bao euinmulus ban quanli trung lii dAy dOc va di\\ dan) mm dugc su dyng cho cac nghien cim niiin III vilio Hep then
Nuoi ll)i)nh Ihyc t n i n g trong ong nghifm Pliumig phap nuiii ihiinh thuc Irimg dugc cai ticn dga tren phumig phap eua Monicn.i \a cs (2011),
Trimg de (2-5 trimg) dui». nuoi in vilro trong 50 pi m6i truong TC199 ( U \1-1W. Earle's salt with L-Glutninine and NaHCOi. Sigma, USA) co bo sung LH (5 gg ml). Estradiol (1 pg/ml), Gcntamycin (50 pg/ml). luiNci thanh bao thai be (10% v/v), Nalri Pyruvate |(l 2'5niM) va FSH vc'n cac nong dg khdc nhau (0.05: 0.1: 0,2 pg/ml)
Cac gigt mot trutmg dugc phii dau khoang va chira trong dla NUNC 35inin, Toan bg dTa nuoi co chira moi truong dugc can bang it nhat 02 h trong tu nuoi Sanyo (Nhat Ban) o dieu kiijn 5%C02, 39°C. dg am bao hoa. Khi hoa long CO: va Ni sir dyng cho cac thi nghiem dugc cung c:ip boi hSng Messer (Singapore).
Trimg nuoi trong moi truang noi tren va chia ra hai 16 cho moi dgt thi nghi?m. Lo 01 nuoi trong tu nuoi Sanyo-1 6 dilu ki?n 5%C02 va 20%O2, 39°C, dg ^m bao hoa trong 32 h Lo 2 nuoi trong tii nuoi Sanyo-2 a dilu ki?n 5%C02 va 5%02, 39''C, dp km bao hoa trong 32 h.
D^nh gia sir thanh thuc cua trirng de Tning sau then gian nuoi thanh thyc a cac dieu kien noi tren dugc ehuyen sang moi truong TCM-
199 Hepes CO bo sung enzym Hyaluromdase (Sigma) a nong dp 1,0 mg/ml, Vi?c phan tach te bao cumulus cua trimg de dugc thuc hi?n trfin kinh hien vi soi noi Nikon SMZIOOO co ban 5m Tokai Hit (37''C) trong 120-180 giayva cok^t hgp phuong phap tach c o h p c 242
Nguyen Mftu Due & T n i n Thi Binh Nguyen (hill len xuong bang pipel thiiy tinh eo ducmg kinh irong lii 130-150 gm).
liimg dugc diinh gia lu Ih^nh ihi^c khi quan sal Ih^iy Ihc eye thir iihal \ii.'il hiC'n (quan s;il tryc tiep hiv'ic nluipm Orcein (l.l".i).
\ i i l> so livu
Cac gi^ Irj dugc irinh bay dudri dgng M + SD, So sanh sg khac nhau ciia hai gia tri trung binh dugc Ihgc hi^n h5ng him /'-lesl v^ 7-icsi, Sg khac nhau giiia cAc .so livu so sdnh dugc xem la co y nghta Ibniigkckhi gialri P - 0.05.
Kl I (,IUA V A I H A O l . U A N Anh hu-mig eiiii nnng dp I SII
Irirng trong cdc nang thugc buong irimg deu it trgng thai chua thinh Ihyc (Hinh 1). Thu ining bilng phwong phap cM lap, cac tning dugc giai phong khoi cac nang trimg (kich iluroc nang tning deu nho hon hojc bang 3mm), Cac tning co chat lugng khac nhau, chi nhihig trimg co chat lugng lot mcri duoc dua vao nuoi in vitro.
c a n cit vao trgng thai lop te bao cumulus bao quanh trimg, ket hgp quan sat trgng thai nguyen sinh chat tren kinh hien vi sol noi Nikon SMZIOOO, tien hanh chgn Igc nhOng trimg co chat lugng tot (lop le bao cumulus bao quanh tning la day djc. it nhat co tir 4 lop tra len) de dimg cho cac nghien ciru E)ieu nay cho phep vi?c nuoi dianh ihuc trimg la kha thi \'a tranh nhimg tieu ton ve hoa chat khi sir dgng cac Irimg CO chat lugng keni
Theo nhi^u tac gia, khi nghien ciru nuoi thanh ihyc uirng de trong ong nghiem thi moi trirong nen chii yeu de qua trinh thanh digc dien ra la moi truong TCM-199 CO b6 sung cac hocmon (FSH, LH, Estradiol-17p) (Baldassarre el ai. 20114. AnguiU el ai, 2007; Paramio. 2010; Momena et al, 2011).
Trong cac nghien ciru, chiing titi cung sii dgng moi truang TCM-199 lam moi truong nen de nuoi thanh thgc trimg de trong ong nghi?m.
Moi truong TCM-199 dugc san xudt bai congty Sigma Aldrich (MJ). khi v ^ ehuyen can bao quan trong dieu ki?n Ignh. Trong thanh phan cua moi truong CO bo sung L-Glutamine. la mpt chat rat can thiet trong hogt dgng song ciia te bao. Neu bao quan khong dung yeu cau va sii dung qua han ghi iren nhan thi L-Glutamine se bi phan huy, anh huong chat lugng moi trudng nuoi rat ro r|t,
Truac khi dua tning vao nu6i trong moi truong TCM-199 da b6 sung cac chat nhir tren da d i cap, do
Tgp chi Cong nghe Sinh hoc 10(2): 241 -245, 2012 la phai phii dau khoang len b^ mgt moi truong va can bJing moi truong it nhk 01-02 h Irong lii nuoi (incubator) co thanh ph^n khi (5'l»CO.i 2()"i.(), hiiac 5%C02+5%02) va nhiet dg xac djnh (39°C). I.irp dSu khoang giiip bao ve moi iruong lianh nhCmg li\c nhan nhiem khuan co thS d^n lir ben ngoai moi truang cung nhu ngan can sg b6c hoi eiia moi trucfng. Viec can bang mgl thai gian nluit dinh moi truong trong tiJ nuoi la c3n thii^l vi d6 chinh l;i ihiri gian dieu chinh pH ciia moi trumig (thong qua lir(,Tng CO2 CO trong til nuoi) cung nhu lam am 11161 Iruimg.
Trong CO the. FSH co anh huong dumig tinh den su phat men eiia tning trong nang trimg. Trong mini cay in vilro, luon luon trong 11161 truang nuoi ihanh
Ihyc irimg phiii c n s u h i c n dien ciia FSH (Keskinlcpe cl ai. 1994; Han el ai, 2008), Day dugc xem la lh;\nh phfin quan irpng va c6 iinh hiroiig dinmg linh den sg phiil trien cua trirng trong qua trinh nuiii Ihanh Ihgc in vilro.
Nong dg FSH sir dyng la bao nhieu thi mire dy anh hmrng cua no dirge xem la co hivu qua iilial. Do la ejiu hoi dugc chiing loi dai ra va Iga chgn ba mifc luSng dg (0,05; 0,1 va 0,2 gg/ml) cua FSH di- khiio sdt trong nghien ciru nay.
Ket i|uii nuoi thanh ihge trirng de trong moi 11 iiinig 1 ( M 199 CO bo sung ciic ehiil can ihiet dugc trinh b.i> n(Baiig 1),
Bang 1 TMethanh thgc ciia tn>'ngd6nu6i trong mfti trudng TCM-199 vdi cac n i n g d$ FSH khSc nhau.
NSng d 9 FSH (0,05pg/inl)
Lo So Sd Tn>ng
thi tn>ng t r i r n g ,hanh nghiem si> thanh
di^ng thuc (n) (n)
t h g c
(V
N&ng d ^ FSH (0,lMg/ml) S6
s v d g n g (n) 22 35 36 33 18 21 165
Sd t r i r n g t h ^ n h thgc (n) 13 25 19 18 8 10 93
Tri>ng t h ^ n h thgc
(%)
59,09 71,43 52,78 54,55 44,44 47,62 54,98 +
N o n g d d F S H ( 0 , 2 M g / m l ) Sd So t r i r n g trirng sir thanh dung thuc (n) (n)
T r i r n g thdnh thgc
(%)
1
Tong
20 10 50
36,11 40,74
11 63,64
60,00 58,33 60,61 50,00 58,82 58,57 + 4,59"
Ghi c h u : " "Gia tri khac nhau co y nghia thong ke (P<0,05)
Ket qua 6 bang I cho thay ti le trimg de thanh thuc (Hinh. 2) chi dat 39,53 + 9.13"o 6 16 nuoi co nong do FSH la 0.05 gg/ml, ti le nay kha thap Khi n6ng dp FSH lang len (0,1 va 0,2 pg/ml) thi ti le thanh thuc (54,98 ± 9,57% va 58,57 + 4,59) dugc cai thien rS ret (P<0,05). Ti le trimg thanh thuc trong moi h-uong TCM199 vai n6ng do FSH la 0,1 va 0,2 gg/ml la khac nhau_(54,98 + 9,57 va 58,57 + 4,59) khong CO y nghTa thong ke (P>0,05).
Ti le trimg thanh thuc trong ong nghiem ciia chiing toi dat tuong duong vai nhom lac gia Moname va dong tac gia (2011). Cac tac gia nay Ihu dugc trimg de o trgng thai thanh thuc trong nuoi cay in vitro voi ti le dao dgng 60%-66%
Voi ket qua trinh bay a bang 1 va so sanh vai nghien ciru ciia Moname et ai (2011), chiing loi Iga chon moi truong TCM-199 co b6 sung LH (5 pg/ml). Estradiol (I pg/ml), Gcntamycin
(50 pg/ml), huyet thanh bao thai be (10% v/v), Natn Pyruvate (0,25mM) va FSH o nong dg 0,1 pg/ml lam moi truong chuan trong nuoi thanh thuc tning de de thuc hien nghien ciiu cai lien tiep theo lien quan nong dp va thanh phan khi trong tit nuoi.
Anh hvong ciia nong do Oxy
Qua trinh phat tnen in vitro ciia trimg de dugc dien ra trong moi truong nuoi nhu da de cap ben tren. Toan bp dTa nuoi dugc dat trong tii nuoi co dieu chinh nhiet dp va nong do khi CO2, Thong thuong, nong dp khi CO2 ciia tu nuoi dugc dat la 5% Day la nong dp thich hgp, tgo dieu kien cho trimg phat tri^n
;;; vilro 6 di^u kien thSp oxy (5%C02 + 5%02), ciing CO the thu dugc cac trimg thanh thuc /// vitro
Ket qua so sanh kha nang thanh thgc ciia trimg de nuoi o hai moi truong nuoi co nong dg oxy khac nhau dugc trinh bay a (Bang 2)
243
Nguyen H&u Dire & Trin Thi Binh Nguy&i
BAng 2. TI 1$ thanh thuc cua Irung d6 nufll trong hai mfll Inrfi-ng c6 nAng d^ oxy khAc nhau
Ldthi nghiem 1 2 3 4 T6ng Ghi chii: '
Dl4u klOn S%CO,-•'20%O, SA trirng S6 tru'ng sCr dgng (n) Ih^nh thgc (n) 21 11 32
24 25 102
19 16 15 61
" G i a m khdc nhau c6 y
Trirng t h i n h thgc (%) 52,38 58.38 66,67 60,00 59,61 i 5.84' nghta l h 4 n g k 6 { P < 0 , 0 5 ) ,
OlAu hl4n 5%COi + S6 trirng
• i > d g n 0 (n) 32 28 19 36 165
5 % 0 , S6 trirng t h i n h thgc (n) 28 24 11 29 93
Trimg th&nh thgc (%) 87,50 85.71 57.89 80.56 77.92 + 13.67*
Kel qua n bang 2 cho iliav. li li; Irimg phdl InOii den giai dogn thanh thgc n lii irung nuoi trong dicu kicn thSp o\v (V'„( ().•'.".,(),) la cao han (P- 0,05) so \ a i 16 tning nuoi trong dicu kicn 5"ii{ ()• i 20"uO-.
Vi^'C nuoi Irimg a dic*u kivn thap O; la khii ton kem. dau lien phai co mgt lii nuoi en c i ehiic nSng dieu chinh \.i hien Ihi nnng dii ( ()- va ca O;, liep d6 phai hg thap O^ bang each sir dung khi Nito. Day chinh la mpt can tra lan khi tnen khai thi nghiem
Tuy nhien. trong nhung truong hgp can ihiet phai CO dugc cac trimg thanh thgc vai hi^u qua cao ban (diing cho muc dich tao phoi nhan ban, ehuyen gen,. .) thi viec su dung mo hinh thap O2 van la mgl giai phap CO the tinh den.
Kel qua ciia chiing toi phii hgp voi cong bo ciia tac gia S i n ^ va dong tac gia (2009) Cac tac gia nay nhan thay, khi nong dp O2 giam xuong con 10% thi hieu qua thu trimg thanh thuc la cao hon nong dp O2 la 20% (P<0.05). Dicu nay mgl lan nC^a cho thay vi?c duy tri nong dg O; thap trong in vitro la co Igi cho sg phat tnen ciia cac te bao, trimg va phoi tao ra sau nay.
Hinh 1. Trtrng d6 tru-fi-c nu6i thanh thyc
Hinh 2. Tr^ng 66 sau nu6i th^nh Ihyc xuit hi^n Itife (Polar Body) vS dia Metaphase II.
Ti le trimg de thanh thgc trong moi truong TCM-199 CO bo sung FSH a n6ng dp 0,1 va 0,2 pg/ml (54.98 + 9.57 % va 58.57 + 4,59 %) cao hon (P<0.05) so \oi moi truong co FSH a n6ng dp 0,05 Mg/ml (39.53+ 9.13" ..)-
Ti 1^ trimg de thanh thgc trong moi tniimg TCM-199 CO bo sung FSH a n6ng do 0,1 pg/ml, 5"uC-0:t5"uO:. 3 9 X (77.92 t 13,67 ",,) cao hon (P- 0.05) so veil nuoi trong moi truang TCM-199 co bo sung FSH o n6ng dp 0,1 pg/ml. 5"oCO;-20?iO., 39°C (59.61 i 5 . S 4 % ) .
L(ri cant on: Nghien cuu ndv duac thuc hien vai sii Idt tr^ kinh phi tir de lai cap Truang Dai hoc Nong nghii'p Nil Noi (ma sd TTRIG2009-12-54)
TAI LIEU THAM KHAO
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Momena Kliatun, Molianiniad Miishiral'Uddin Bhuiyan, Jalal Uddin Ahmed. Aminul Haqiic, Mohammad Do/lui Rahman. Mohainincd Shanisuddin (2011) In vino maturation and ferlili/alion ol piepubenal black IJcngal goal oocytes ./I'd Sa 12(1) 78-82,
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E F F E C T S O F F O L L I C U L A R S T I M U L A T I N G H O R M O N E A N D O X Y G E N C O N C E N T R A T I O N ON T H E IN V I T R O M A T U R A T I O N O F C A R P R I N E O O C Y T E Ngu> en Huu Due . Tran Thj Binh Nyuj en
Hanoi Univer":!!}- of Agriculture
SUMMARY
This study was conducted for evaluating the effects of FSH hormone and oxygen concentration on the proportion of in vitro matured caprine oocytes. Experiment I: caprine oocytes were recovered from ovaries and in vitro cultured for 32 hrs in TC199 medium (Earle's salt widi L-Glutamme and NaHC03, Sigma, USA) supplemented with LH (5 pg/ml). Estradiol (i pg/ml), Genlamycin (50 tig/ml). Fetal Bovine Serum (10% v/v), Natri Pyruvate (0,25mM) and FSH at different concentrations (0 05; 0 1, 0 2 pg/ml), S%C02, 39°C.
Experiment 2: Capnne oocytes were cultured for 32 hrs in TCM-199 supplemented with FSH 0.1 fig/ml and cultured in two incubators: Isl incubator (5%CO2-(-20%O2, 39°C) and 2nd incubator (5%C02+5%O2, 39°C).
Results indicated ihai proportion of matured oocytes m TCM-199 supplemented with FSH at 0 I va 0.2 pg/ml (54 98 + 9 5 7 % and 58.57-I-4.59%) was higher (P<0,05) than those of FSH al 0,05 jig/ml (39.53-t-9 13%);
proportion of matured oocyte in TCM-199 supplemented with FSH at 0,1 ng/mi, 5%C02-^5%02, 39'C (77.92 + 13.67%)washigher(P<0,05) than those ofFSH at 0,1 pg/ml, 5%CO2+20%O2, 39''C (5961 +5.84%) In conclusion, matured caprine oocytes were obtained by in vitro maturation method in Vietnam, they can be used for in vitro caprine embryo production via in vitro fertilization or cloning technique.
KeywordszCapi ine oocyte, concentration, follicular stimulating hormone, in vilro maturation, oxygen
Author for correspondence: Te]. +84-1699606099, E-mail: nhduc&.hua.edu.v.