Tap chi Cdng nghe Sinh hoc 11(2): 225-234, 2013
A N H H U O N G C U A T E B A O B I E U M O O N G D A N T R L N G D E N S U P H A T T R I E N /.V VITRO C L A P H O I B O T H L T I N H T R O N G O N G N G H I E M
Nguyin Hfru Dire', T r 5 n Thj Binh N g u y e n \ Le Thj Chau^
Tnr&ng Dai hgc Nong nghiep Hd Not
Vi^n Nghien ciru khoa hgc Tdy Nguyen, Vien Hdn ldm Khoa hoc vd Cong nghe Viet Nam
TOM TAT
Nghien cuu nay dugc ti^n hanh nham danh gia anh buong ciia viec bo sung t% bao 6ng dan tning trong moi trudng nuoi den su phat tnen m vuro cila phoi bo thu tinh trong 6ng nghiem. TS bao biSu mo 6ng din tning duoc phan lap theo phuong phap ciia Waller (1995), co cai tien Chimg dugc nuoi ciy theo phuong phap cua Kim va dong tac gia (2011), co cai tien, trong tii nuoi Sanyo o nhiet do 37°C, 5% CO,, do am bao hoa Moi truang nuoi te bao la moi truong Dulbecco's Modified Eagle Medium (D"MEM) co b6 sung 10% huyit thanh bao thai be, b6 sung khang smh Penicilm 100 000 Ul/lit va Streptomycin 0,05 gr/lit Lop don t^ bao ong dan trimg dugc tao ra dua tren phuong phap cua McGowan (2011) co cai nin. Tning duoc nuoi thanh thuc theo phuang phap ciia Due va dong tac gia (2003). Chgn loc tmh triing khoe, co chat luong t6t bang phuong phap boi nguoc (Swim-up) theo phuong phap ciia Parrish va dong tac gia (1986b), co cai tien trong raoi trucmg Brackett-Ohphant (BO) Sau cac khoang thai gian khac nhau (20, 30, 40, 50, 60, 70, 80 phiit) thu nhan va xac dinh nong dg tinh triing khoe bang may Spermacue. Thu linh trong ong nghiem giiia trimg bo da thanh thuc va tinh triing chgn lgc diln ra trong tii nuoi Sanyo o dieu kien 5% CO2, 39°C, do am bao hoa theo phuang phap ctia Dire va dong tac gia (2003) co cai tien M61 tmong thy tmh la moi truong BO co bo sung 1,0 |iM Epincpherine, 10 pM Hypotaurine, 20 pM Penicillamine, 10 ng/ml Heparme Phoi duoc nuoi trong moi truang TC199 bo sung 10% huyet thanh bao thai be, c6 va khong co bo sung te bao bieu mo ong dan trimg. Ket qua cho thay 1/ Sau 60 phiit bai nguac, linh triing van dgng tien thang co chat luong tot duoc thu nhan la dat vc so lugng de diing cho thu nnh ong nghiem, 2/ Ti I? phoi phan chia a giai do^n 2-4 te bao va 4-8 le bao lan lugt a 16 khong va co bo sung te bao ong dan trimg la khong khac nhau (P>0,05), lan luat la 69,39% so vai 70,34%
va 54,71% so vai 53,61%, 3/Ti le phoi dau-phoi nang a 16 khong bo sung te bao ong dan trimg thap han c6 y nghTa thong ke (P<0,01) so vai lo co bo sung te bao, lan luat la 17.20% so voi 30,34% Ket luan te bao bieu mo ong dan trirng co anh huang duong tinh den sg phat tnen ciia phoi bo thu lmh trong ong nghiem Tirkhoa BOECs, nuoi cd\. phoi, thu iinli. in vitro, tlianh iliiu, innig, lmh iriing
M O D A U Cong nghe phoi (Embryo Biotechnology) dong vai tro quan trpng trong cong nghe smh hgc sinh san Cong nghe sinh hoc sinh san dgng vat (Animal noi tren Cong nghe nay da va dang duoc thuchien Reproductive Biotechnology) ngay nay dugc hi^u tai nhieu nuoc co nen chan nuoi lien lien cua the gioi nhu mot to hgp ciia cac ky thuat sinh san, di truyen, (My. Nhal Ban, Diic. Phap, Tmng Quoc. ) Cho den smh hgc lc bao va phan lu nham sit dung hgp ly cac nay, cong nghe phoi bo da co nhung buac tien dang ngu6n nguyen ligu sinh hoc mang thong tm di truygn ke tir khi be ihu tinh ong nghiem ra doi tai My vao (tinh trung, tning, t^ bao smh duong. ). nam 1981 (Bracked fi « / , 1982).
Cac ngi dung co ban ciia cong nghe sinh hoc sinh NhCrng nam tiep theo, tai nhieu phong thi nghiem san bao g6m, cong nghe gay dong due d6ng loat, tren the gi6i, cac ky thuat hen quan khong ngirng cong ngh? gay rung trimg nhidu, ky thuat thu va cSy dugc nghien ciru cai lien: nuoi trimg thanh thuc phoi, dong lanh phoi, thu tinh trong 6ng nghiem, irong ong nghiem (in vilro mamralion), thu tmh ong khai thac irurng vai su trg giiip ciia may sieu am nghiem (in vi/ro fertilization) va nuoi phoi trong ong {Ovum Pick-Up), cat phoi, nhan dong phoi tir l8 bao nghiem (in vitro culture) (Fukui et al, 1983, Iriiani phoi, xac djnh gioi linh ciia phoi bing PCR, lach tinh et al, 1984. Criser et ai. 1984, Fukushima et al.
triing duc/cai, tao phoi nhan ban bang ky thuat cdy 1985, Fukuda et al, 1990; Fukui et al, 1991. Bols et nhan ti bao sinh du&ng (Somatic Cell Nuclear a/., 1995, Galli c; a/,, 2001, Oyamada e / a / , 2004, Transfer), chuyen gen... O i k a w a e r a / . 2005; Shirayi et cd., 2009).
Nguyen HiJu Ditc et al.
Ngoai viec nuoi chung (Co-culture) phoi thu tinh trong ong nghiem voi te bao bieu mo ong dan trirng bo (Bovine Oviduct Epithelial Cells), cac phong thi nghiem con nuoi chung voi cac ioai te bao khac nhu tk bao MDBK (Madin Darfay Bovine Kidney), BRL (Buffalo Rat Liver), Vero (Cellules de rein de singe vert), te bao Granulosa, te bao cumulus (Aoyagi et ai, 1990; Fukui c/a/., 1991, Carolan ^r ti/,, 1995;
Lima era/., 2004; Shirazie( a/., 2009).
Tai Viet Nam, cac nghien cuu ve tao phoi bo trong ong nghiem cdng da dugc lien hanh, tuy nhien, viec xay dung va chirng minh hieu qua ciia viec nuoi cay chung te bao bieu mo ong dan trimg voi phoi ihu tinh ong nghiem con chua dugc quan lam.
Do vay, de liep tuc nang cao hieu qua tao phoi ho trong ong nghiem, chung toi thuc hien nghien ciru nay voi muc lieu la xay dung va danh gia hieu qua ciia viec nuoi cay chung (co-culture) te bao bieu mo ong dan trirng va phoi thu tinh ong nghiem trong dieu kien cu the ctia Phong thi nghiem Cong nghe sinh hgc dgng vat, Khoa Cong nghe sinh hgc, Truong Dai hoc Nong nghiep Ha Noi
VAT LIEU VA PHU'ONG PHAP Hoa chat, dung cu, t r a n g thiCt bi
Tat ca boa chat, moi truong su dung trong nghien ctru nay deu dugc cung cap boi hang hoa chSt Sigma (My), Gibco (Anh), khi CO2 hoa long cua hang Messer (Singapore), dia nuoi te bao, dia thu tinh ong nghiem dugc cung cap boi hang NUNC (Dan Mach): cong ra dong lanh dugc cung c§p boi hang IMV (Phap) Thiet bi van chuyen mdu chuyen dung Cryologic (Uc), tii nuoi te bao va phoi Sanyo (Nhai Ban), kinh hign vi phan pha Nikon Ti-U (Nhat Ban), kinh hien vi soi noi Nikon SMZIOOO, ban ^m Tokai Hit (Nhat Ban), may ly tam Hettich (Dire), may lam am 6ng nghiem Grant (Anh), may d^m tinh trimg tu dpng SpermaCue (Dire), bu6ng cdy vo triing Labconco (My)
Thu buong t r u n g b6
Bu6ng trirng fao duoc thu thap lai 16 m6, bu6ng irirng duoc cat ngay khi con vat vira moi chgt va duoc rua lai 03 lan bang nuoc mu6i sinh ly NaCI 0,9% am (37°C) co bo sung khang sinh Penicilm (10000 Ul./lil), Streptomycin (0,05 g/l). Ti^p theo, chiing duoc bao quan trong ihi^I bi van chuyen mau chuyen dung Cryologic dat 6 37''C va mang v&
phong thi nghiem trong vong 2 - 4 h.
K h a i t h a c t r i m g tir buong t r i m g
Buong injng dugc nia lai 02 lan bang dung dich Phosphate Buffered Saline (PBS) km {iTC) co thanh phan (dimg de pha 01 I) gom NaCI (8,0 g), KCl {0,2 g), Na:HP04 (1,15 g), KH2PO4 (0,2 g).
Glucose (1,0 g), CaCl2.2H20 (0,132 g), MgCl2.7H20 (0,121 g), Natri Pyruvate (0,036 g), Penicilm (10000 UI/1), Streptomycin (0,05 g/l), pH 7,3-7,4.
Trimg trong cac nang duoc thu bang each cat lop buong tning (dimg dao mo cat thanh cac duong song song tren khap be mat buong tning), Khi cat, buong trimg dugc ngam trong moi trucmg TC-I99 Hepes (Sigma). Trung dugc giai phong khoi buong tning dugc soi tim Iren kinh hien vi soi noi Nikon SMZIOOO,
Qua trinh soi tim trirng tren kinh hien vi dugc lien hanh trong buong cay vo triing Labconco (My) va Iren kinh hien vi luon luon co ban am 37°C Tokai Hit (Nhat Ban). Chi nhiing trimg co chat lugng lot (co it nhat 04 lop te bao cumulus bao quanh triirng, cac lop te bao nay day dan, cac te bao lien ket chat che) moi duoc su dung cho cac nghien ciiu nuoi in vhro liep theo.
Phan lap te bao bieu mo ong d a n trirng Te bao bieu mo ong dan tning duoc phan lap bang phuang phap cao (scrape) theo Walter (1995), CO cai lien, cu the nhu sau Ong dan tning dugc cat ra va duoc rua lai 02 lan Irong moi truong PBS moi (37°C). Co djnh ong dan trirng len lam kinh. Diing keo mo cat dgc ong dan trirng voi muc dich bgc lp lop bieu mo ben trong cua ong dan tning. Sir dung dao mo cao nhe nhang be mat ben trong ciia ong dan Ining de tach nhirng mang te bao bieu mo
Cho nhirng mang te bao bieu mo nay vao ong ly tam co chira 10 ml moi truong Dulbecco's Modified Eagle Medium ( D ' M E M ) c 6 b6 sung 10%
huyel thanh bao thai be, b6 sung khang sinh Penicilline 10()000 Ul/1 va Slreptomycine 0,05 gr/lit. Ly lam toe dp 1200 vong/phiil trong 05 phut, giir nguyen lop te bao lang dgng ben duai, loai bo dich noi ben tren. Diing pipet Pasteur vo triing hiit moi Iruong co Iron ISn t^ bao len xu6ng 08-10 lan de Iron deu. Them den lOml moi truang D'MEM CO bo sung 10% huyet thanh bao thai be Lap I91 qua trinh ly tam 04 lan. Thu nhan ti bao bieu mo ong dan trirng dang mang nho.
Quan sat cac mang te bao bieu mo ong din trung tren kinh hien vi phan pha Nikon Ti-U
Tap chi Cdng nghi Sinh hgc 11(2): 225-234, 2013 Nuoi caj trong ong nghiem t l bao b i l u mo 6ng d a n t r i m g
Cac mang le bao bieu mo ong dan tning sau phan lap dugc nuoi cay theo phuang phap cua Kim va dong lac gia (2011), c6 cai lien, cu the nhu sau:
Chuan bi lii nuoi Sanyo 6 che do can bang: nhiel dg 37''C, 5% CO2, dp am bao hoa. Diing dia nhua NUNC (loai 04 gieng), cho 1,5 ml moi truong D'MEM CO bo sung 10% huyit thanh bao thai be, b6 sung khang sinh Penicilin 100.000 UI/l va Streptomycin 0,05 g/l vao moi gilng. Phii len b^ mat 1,5 ml moi truang noi Iren 0,5 ml dau khoang (mineral oil), day nap, can bang khi va nhiet do trong til nuoi trong il nhal 02 h.
Cac mang te bao bieu mo ong dan trimg vira dupc phan lap dugc tien hanh nuoi cay trong moi gieng noi tren a diSu kien nhiet dp 37"C, 5% CO2, do am bao hoa. Quan sat su phat trien in vitro ciia cac te bao bieu mo ong dan tning tren kinh hien vi phan pha Nikon Ti-U
Tao lop doTi t§ bao bieu mo ong dan trirng Lap dan te bao ong dan trimg dugc tao ra dua tren phuang phap ciia McGowan (2011) co cai lien, cu the nhu sau: Sau 48 h nuoi cay, dimg Trypsin 0,1%
tach lop te bao bieu mo ong din trimg khoi be mat day cua dTa nuoi Thu lay te bao, cho vao ong ly lam co chira moi truang nuoi cay. Ly lam loc do 1200 vong/phiil. Hut bo moi truang ben tren, hoa tan nhe nhang lap te bao duai day bang each su dung pipet Pasteur. Them moi truang nuoi cay va lap lai 02 lan ly tam-lhay moi truang nhu vira irinh bay
Gieo te bao bieu mo ong dan trirng vao dia NUNC mai (loai 04 giSng) vgi nong do IxlO" te bao/ml Trong dia nuoi c6 chiia sin moi truang D'MEM CO b6 sung 10% huyet thanh bao thai be, bo sung khang sinh Penicilm lOOOOOUI/1 va Streplomycin 0,05 g/1.
Sau 24 h nuoi cay, te bao bieu mo ong dan trimg tao Idrp dan bam duai day dTa nuoi. Thay moi truong nuoi t l bao bang moi truang nuoi phoi (TCM199 bo sung 10% huyll thanh bao thai be) de chuan bi nuoi phoi hb thy linh ong nghiem.
Nuoi thanh thuc t r u n g trong ong nghiem Phuang phap nuoi thanh thuc trirng duac cai lien d\ra tren phuang phap cua Dire va dong tac gia (2003), c^ ih^ nhu sau: Chuin bj moi truong TCI99 (TCM-199, Earle's salt wilh L-Glulamine and
NaHCOj, Sigma, USA) co b6 sung LH (5 p g m l ) . Estradiol (I pg/ml), Gentamycin (50 p g m l ) . hu\et thanh bao thai be (10% v/v), Natri Pyni\ate (0.25 mM) va FSH (0,1 pg/ml). Lpc moi truong qua mang loc Millipore co kich thuac 16 0,22 pm.
Toan bg dTa nuoi (loai 04 gieng) co chiia moi truang duoc can bang il nhdt 02 h trong tii nuoi Sanyo (Nhat Ban) a di^u kien 5%C0:, 39''C, do dm bao hoa.
Tning bo dupc cho vao dTa nuoi (20-25 Irimg/gieng) co moi trucmg da chuan bi 6 tren va qua trinh thanh thuc xay ra trong 24 h a dieu kien 5%
COi, 39°C, do am bao hoa,
Chon loc tinh trung dung cho thu tmh trong ong nghiem
Tinh triing dong lanh ciia bo Holslein dugc su dung de thu linh ong nghiem voi trirng thanh thuc da noi o tren Tinh triing dugc giai dong bang each lay nhanh cong ra (Ioai 0,25ml) khoi Nita long roi nhiing cgng ra vao coc nuac 37''C trong vong 0! phiit,
Sau do tmh trimg giai dong dugc bom vao day mot ong nghiem nho va nhe nhang phii len tren do 02 ml moi truang BO co thanh phan nhu sau (diing pha 01 1) gflm NaCI (6,55 g), KCl (0,3 g), CaCl2 2H20 (0,33 g), NaH^PO, H.O (0,113 g), MgClj.bHjO (0,106 g), N a H c b 3 (3,104 g). Glucose (3,5 g), Natri Pyruvate (0,137 g),BSA (3,0 gr).
Chon lgc tinh Iriing van dong lien thang, co chat lugng lot bang phuang phap boi nguoc (Swim-up) theo phuang phap ciia Parrish va d6ng tac gia (1986 b), CO cai tien, cu the nhu sau: dat ong nghiem noi tren nghieng mpl goc 45*^ vao trong tu nu6i co 5%
CO2, 39''C, dp am bao hoa, Sau cac khoang thai gian khac nhau 20, 30, 40, 50, 60, 70. 80 phiit, cdn than lay ong nghiem ra va hiit 0,5 ml moi truang ben tren (co chira tinh triing van d6ng lien thang), cho vao ong eppendorf (loai 1,5 ml), day nap, ly tam 1000 vong/phiit trong 05 phut. Hiit b6 m6i truong ben Iren. giir can duai co chira tinh trimg khfle, chat luang t6l. Xac dinh nong do tinh trung khoe thu dupc sau Swim-up bang may Spermacue Thu tinh trong ong nghiem va nuoi phoi giai doan S6TO
Trimg bo sau nuoi thanh thuc dupc chuyen sang dTa nuo! men de thu tinh voi tinh triing vira chgn Igc Qua trinh thu Unh in vuro dien ra trong tu nuoi Sanyo a dieu kien 5% CO2, 39°C, do am bao hoa theo phuong phap ciia Dire va dong tac gia (2003) co cai tien
Nguyen Huu Due et ai Nong do tinh triing su dung cho thu linh ong
nghiem la 2x10^ linh triing/ml moi truang ihu tinh.
Moi truong thu tinh co thanh phan chii yeu la moi truong BO va c6 b6 sung 1,0 pM Epinepherine, 10 ftM Hypolaurine, 20 pM Penicillamine, 10 pg/ml Hepanne.
Sau 24 h ke tir khi cho trirng va tinh trimg gap nhau trong 6ng nghiem, lay tning ra khoi moi truong thu tinh, chuyen vao dia nuoi co chiia moi truong TC199-Hepes, tach sach lap le bao cumulus bao quanh trirng va chuyen cac tning thu tinh gia dinh vao moi truong nuoi ph6i
Moi truang nuoi phoi la moi tnrdng T C i 9 9 bfl sung 10% huyet thanh bao thai be, co va khong co bo sung te bao ong dan trirng Quan sat va ghi nhan so phoi a giai doan 2-4 le bao, 4-8 t^ bao, phoi dau- phoi nang tren kinh hien vi soi n6i Nikon SMZIOOO c6 ban dm Tokai Hit (37''C),
X u l y so heu
So sanh su khac nhau cua hai gia Iri dugc thuc hien bang ham /-"-test va T-test, Su khac nhau giua
cac so lieu so sanh dugc xem la co y nghTa Ihong ke khi gia tri P < 0,05.
KET QUA VA THAO LUAN Tao lop d o n te bao bi^u m o ong d a n t r i m g
Te bao bieu mo ong dan trimg dugc phan lap, nhan nuoi f;i vitro va tao lop te bao dan. Viec nhan nuoi trong ong nghiem te bao bieu mo ong dan tning cung nhu nhieu loai le bao dugc thuc hien trong tii nuoi chuyen dung a dieu kien 5%C02, 37°C, do dm bao hoa.
Lop te bao don dugc quan sat va danh gia tren kinh hien vi phan pha Ti-U nham truoc het xem xet tinh dong deu, kha nang phat trien cung nhu kha nang bl nhiem khuan truoc khi chuyen thay moi trucmg mm va chuyen phoi thu tinh 6ng nghiem vao. Neu nghi ngo nhiem khuan thi tuyet doi khong su dung.
Sau khi tao duac lap le bao don (Hinh 1), chiing toi lien hanh thay moi truang D'MEM bang moi truang T C I 9 9 de chuan bi chuyen phoi vao sau do mpl ngay
, '<^^-Si^
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Hinh 1, T^ bao bieu rr • ong dan tru-ng phat Inen trong moi Iruong D'MEM, 10% huy§i thanh bao thai be Thanh phan moi truang nuoi cdy t l bag duoc
thiet lap gan giong nhu thanh phdn dich long trong eg the song bao g6m miifli vo ca, carbonhydrate, Miamin, amino acid, cac tien chdt biln duang. nhan i6 lang truong, hormone, cac yeu 16 vi lugng va dac biet quan trong la huyet thanh, Cac chdt dinh duang thiel yeu gnip le bao phan chia nhu amino acid, acid beo, duong, cac ion, cac vitamin, co-factor va cac phan tir can thiet de duy tri moi truong hoa hoc cho cac te bao
Moi truong diing de nuoi cdy t l bao 6ng dan trimg thong ihuong la Eagle Minimal Essential Media (MEM) (Eagle, 1959) hoac Dulbecco's Modified Eagle's media (D'MEM) (Dulbecco va Freeman, 1959) hoac moi tnrdng tdng hgp TCM-199,
Moi tmong Eagle's Minimal Essential Media (MEM) con goi la moi truang tdi ihilu, do Eagle thiel lap. Day la moi trudng ca ban co chira ndng do cac amino acid va vitamin, cung cin b6 sung 5-10%
Tgp chi Cdng nghi Sinh hgc 11(2| 225-234. 2013 huyet thanh khi nuoi cay te bao. Mdi Irudng D'MEM (Dulbecco's Modified Eagle Medium) do Dulbecco cai tien tir mdi trudng E'MEM (Eagle's Mmimal Essential Medium) hien dang dugc sir dung chu yeu trong viec nufli cay te bao, dac biel la te bao gdc.
Nd CO ndng do amino acid cao gap ddi, nong do vitamin cao gap bdn moi truong E'MEM, tdi uu cho nudi cay te bao cd mat dd cao
Chinh vi vay ma D'MEM la mdi Irudng thuang pham thich hap cho nudi cay nhieu ioai le bao dgng vat va chiing toi quyet dinh sir dung moi trudng nay cho viec nudi cay te bao bieu md dng dan trimg.
Huyet thanh duac bd sung vao moi trudng de lao dieu kien tdi uu cho nudi cay te bao Tharlh phan chinh cd trong huyet thanh la protein, ngoai ra nd cdnchira nhieu polypeptid,cac nhan to tang trudng, li pid, hormone, amino acid, glucose, keloacid va cac nguyen td vi lugng nhu sat, ddng, kem. , rat can thiet cho su phal trien ciia le bao.
Chon loc t i n h triing diing t r o n g thu t m h ong nghiem
Tinh triing ddng lanh ciia cac due gidng Holstein dugc giai ddng va chpn lpc cac linh triing cd chat luong tdt diing cho muc dich thu linh trong dng nghiem. Trong moi cpng r?, sd lupng linh triing dao dgng tir 12 tneu - 15 tri?u, trong do 25-35% la nhirng tinh triing cd chat lugng tdt, dat yeu cau diing cho thu tinh dng nghiem
Sau nhCtng khoang thai gian khac nhau de tinh triing lu do boi ngugc len trong dng nghiem, chiing Idl thu dugc kel qua Irinh bay d bang 1, Bang 1. Kha nSng thu nhpn tmh triing chat lugng t6l diing cho thg tmh Ang nghi#m thong qua phuong phap boi ngugc (Swim-up)
Tho'i gian xi> ly (phut)
S6 lifong tinh triing tot thu nhan (Min-Max, trieu/ml) 0,10-0,22 0,23 - 0,35 0.67-0,78 0.95-1,25 1,45-2,23 2,35-2,64 2.58 - 3,02
Kit qua d bang 1 cho thdy, tir 20 den 60 phut bai ngupc, sd lugng tinh trimg co chat lugng tot thu nhan \k khi Ihdp, chua du cho mgc dich thu tmh ong
nghiem. Sau 60 phiit boi ngugc, sd lugng tinh triing chat luong tot thu nhan la dap img vgi yeu cau, cac tinh Iriing nay van ddng lien thang, hoan loan khong cd hinh dang bat thudng.
Hien nay, cd hai phuong phap phd bien uong chpn loc tinh triing diing cho muc dich thu tinh uong ong nghiem, do la phucmg phap bai nguac (Swim-up) va phuong phap lpc qua phan Idp PercoII. Hieu qua thu tinh dng nghiem cua tinh triing sau khi xir ly bang hai phuang phap nay la khdng khac nhau Trong mot nghien ciiu cua Seidei va d6ng lac gia (1995), khi nghien cim ve van de tren, cac tac gia danh gia su phan chia cua phdi d thdi diem 42 h sau thu tinh. Ket qua cho thay, d 16 tinh triing xir ly bang phuang phap lgc qua phan idp PercoII, li le phfli phan chia dal 68%, kel qua nay khdng khac ve mat thong ke so vdi Id linh triing xir ly bang phuang phap boi ngupc (dat 71 %)
Ndng do tinh triing dua vao mdi trudng Ihu linh cung la mdt yeu td c6 anh hudng den ket qua tao phoi. Th6ng thudng, ndng dg nay dao dong trong khoang lir IxlO'' den 2x10* tinh triing/ml. Neu ndng do thdp dual nguong nay thi sfl lugng tinh triing thap, lupng enzym giai phong lir the dinh ciia tinh trimg khflng du de pha ldp mang te bao cumulus bao quanh trirng cQng nhu khong dii de xuyen qua lop mang sang va mang te bao chal ciia tning, tir dd dan tdi ti le thu tinh dat duoc rat thap. Tuy nhien, neu nong dd tinh trimg cao hon ngudng tren thi lai thay xual hien hien tuong da thu tmh, lire la nhieu linh triing dl vao moi triing (hien tugng polyspermy), dieu nay lam ket qua thu nhan phdi chat lugng dal chuan ve sau ciing thap. Do vay, trong cac thi nghiem ciia chiing tdi, viec xac dinh ndng do tinh triing dua vao thu linh la can thiet va chiing ldi quyel dinh diing ndng do linh triing trong moi truong thu tinh la 2x10* linh Ining/mL
Mgt luu y khac khi chgn lpc tinh triing la tdc do ly tam, Khi hiit mfli Irudng ben tren dng nghiem cd chira tinh triing khoe sau thai gian boi ngugc, can het sire nhe nhang chuyen chiing vao flng eppendorf de ly tam. T6c dp ly tam hop ly thuong dao ddng trong khoang 1000-1200 vdng/phiit, Neu tdc dg ly tam dudi ngudng nay, kha nang thu nhan tinh triing la khdng Iriei de, tuy nhien, neu vuot nguong nay thi tinh triing lai bi don manh ve day flng ly lam, dieu nay cd anh hudng khdng tot den sire sdng va kha nang thu linh ciia tinh trimg ve sau,
Anh h u o n g ciia te bao bieu mo ong dan trirng den sir phat trien m vitro cua phoi bd
Sau khi thy tinh cac phdi gia dmh duoc chuyen
Nguyen Hiru Diic et al.
vao cac dTa nudi co mdi irudng T C I 9 9 , bd sung 10%
huyet thanh bao thai be, co hoac khflng cd ldp dan te bao bidu md dng dan triing Ket qua sd phdi d giai doan 2-4 t l bao, 4-8 t l bao va phdi dau-phdi nang dugc trinh bay trong bang 2.
Ket qua d bang 2 cho thay phdi thu tinh trong dng nghiem phat tnen lot ban trong mdi trudng cd bd sung t l bao bieu md dng dan tning: ti le phdi d giai doan 2-4 te bao d mdi Irudng T C I 9 9 khdng va CO bfl sung te bao bieu md dng dan Ining lan lupt la 69,39% va 70,34% (P>0,05); ti le phdi d giai doan 4-8 le bao d hai moi trudng noi tren lan luoi la 5 4 , 7 1 % va 5 3 , 6 1 % (P>0,05): nhung trong mfli trudng co bd sung te bao bieu md dng Bang 2. Kha nang phat trien ciia phoi bo thu tmh ong nghiem trong n mo ong dlin trtpng
dSn tning ihi ti le phoi phat tnen den giai doan phdl dau-phdi nang la cao hon cd y nghTa thdng ke (P<0,01), cu t h i trong moi trudng T C I 9 9 co bo sung t l bao b i l u md 6ng dan tning thi li le phdi dau-phdi nang thu d u g c la 30,34%), trong khi do ti le nay d mdi Irudng T C I 9 9 khdng bd sung te bao b i l u md dng dan Iriing chi dat 17,20%i, Ket qua nay phii hgp vdi ket qua ciia mpl sd tac gia tren Ihl gidi khi nghien ciiu van de nay. Cu the, ti Ie phdi dau-phoi nang trong mdi trudng cd bd sung t l bao dng d i n trirng cao han co y nghia thdng ke (dao ddng 22,55%-34,62%) {P<0,01) so vdi kk qua thu nhan trong mdi Irudng khdng bd sung ong ddn ining { I 5 , 7 0 % - I 9 , 3 2 % (Eyeslone, First, 1986;
LuetaL 1987a).
» truong TCI99 co va khong co bo sung te bao bieu
M6i trucFng nuoi phoi T C I 99
TC199+te bao bieu mo ong dan tripng Chiiy- b(P<0,01)
Trtpng (n) 245 236
thanh t h u c Phoi 2-4 te bao
(%)
69,39 (170/245) 70,34 (166/236)
Phoi 4-8 te bao
(%)
54.71 (93/170) 53,61 (89/166)Phdi dau-Phdi nang
(%)
17,20^(16/93) 30,34 "
(27/89)
Cac nghien ciru lai nhieu nudc trong thap ky 60 va 70 cho thay cac phdi bd va ciru dugc lao ra trong CO the (in vivo), sau dd thu lay va dem nudi in vitro thi chiing rat khd co kha nang phat trien vuot qua giai doan 8-16 te bao. Do vay, cac nha nghien ciru da rat no luc trong viec lam giau, bd sung nhieu chat khac nhau vao moi trudng nufli phdi cd dien de cai thien linh hinh tren. Tuy vay, ket qua dat duac la khdng kha quan.
Sang den thap ky 80, vdi su phat hien vai Ird ciia te bao dng dan trimg trong nudi phdi in vitro, tinh hinh tren da dugc cai ihien mdt each co ban. Nhieu bao cao cua cac nha khoa hgc cho thay viec nuoi chung (co-culture) te bao dng dan tning va hpp tu cung nhu phoi giai doan sdm (2-4 te bao) cho phep thu dugc cac phdi d giai doan phdi dau/phdi nang khdng chi ddi vdi bd ma cdn tren nhieu ddi lugng ddng vat khac (Bd Eyestone , FirsL 1986, Lu et al.
1987a, Cira: Gandolfi et al. 1986; Ngua, Battul ei al. 1990, Thfl; Carney et al. 1990; Chupt: Ouhibi et U/..1988. Sakkas, Trouson, 1990; N g u d r Bongso etal. 1994)
Tai Bi, Mermillod va dong tac gia (1991) da lam thi nghiem va chirng minh le bao ong din trimg ciia
thd cd the giiip phdi bd phat tnen. Tuy nhien, hieu qua cua viec thay the le bao dng dan tning khac loai nay (thd) khdng bang sii dung chinh te bao ong dan trimg ciia loai do (bd). Trong giai doan nay, ciing c6 mdt nghien curu dang chii y, dd la sir dung le bao ong dan tning bo bd sung vao moi trudng nudi, co Ih^
giiip phdl ngudi phat trien tdt hgn trudc giai doan cay phoi hoac dong lanh (Wiemerf/ ai, 1993)
Gadolfi va ddng tac gia (1989) la mgt trong nhirng ngudi dau tien chirng minh rang hien tugng
"Block" d giai doan 08 le bao khdng xay ra doi vdi phdl lao ra in vivo, lay ra d giai doan trudc 08 le bao va sau dd dem nudi in vitro trong su cd mat ciia ldp le bao dng ddn tning. Cac tac gia nay da lam viec rat nhieu va hieu qua nham thiet lap mpt he ihong nuoi cay chung te bao dng dan tning va phdi d vao thai diem do
Ong dan trirng bd chinh la mgl vi mdi trudng thich hgp cho qua trinh thanh thuc cudi ciing cua tning, thich hgp cho su thu tinh, ddng thdi dd ciing chinh la vi mdi truong thuan lgi cho su phat tnen ciia phdi giai doan sdm Viec tao ldp te bao dan dng dan tning ma cac nha khoa hgc tien hanh ciing nh5m dimg muc dich tren, dd la tao mgt vi mdi trucmg
Tap chi Cdng nghi Sinh hgc 11(2) 225-234, 2013 thuan lai nhu da noi, chi khac la d diiu kien in vitro ma thdi.
Cac p h u a n g phap dugc su dung d l thu l l bao dng dan Irirng la thay ddi, khac nhau giira cac phdng thi nghiem cung nhu nhdm nghien ciiu Dau Uen, cd the ke den la phuong phap sir dung enzym (Eyestone et ai, 1989; Lu et ai, 1987; Joshi, 1988), ke den cd the de cap phuang phap thut rira don gian dng dan triing ciia dpng vat song (McCaffrey et ai. 1990) hoac hien nay la su dung cac dgng vat d Id md de thu ong dan trirng va tach ldp te bao dng dan trirng tai phdng thi nghiem (Fukui, Ono, 1988; Vergos, 1990). Phuang phap sir dung Trypsin de phan lach va nhan nuoi le bao flng dan tning cua nhieu loai khac nhau dugc m6 ta bdi Ouhibi va dflng tac gia (1988).
Trong cac nghien ciiu ban dau ve viec thu nhan le bao flng dan tning d bfl, cac nha nghien cim dal ra cau hdi la: viec thu nhan te bao nen duac lien hanh vao giai doan nao ciia chu ky ddng duc. NghTa la, lieu CO sy khac nhau hay khdng ve hieu qua lao phoi dau/phdi nang khi thu nhan le bao flng dan trung d pha the vang hoac pha tning rung. Mdt nghien ciru ciia Vergos (1990) da chiing mmh dugc rang trang thai sinh ly cua chu ky dgng duc la khong co gia In quan trgng cho viec thu nhan te bao ong dan tning, Ket qua nghien ciiu ciia Thibodeaux va dong lac gia (1991, 1992b) tai My ciing chi ra rang te bao dng ddn tning thu nhan d giai doan sdm hoac mugn ciia pha the vang cua chu ky dgng due la hoan loan tuang duong nhau ve mat hieu qua khi nudi cay chung vdi phdl bo giai doan sdm.
Tuy nhien, mgl van de ma cac nha khoa hpc phai quan tam, dd la tinh dn dmh d trang thai binh thudng cOa le bao dng ddn trimg khi dugc nudi in vitro trong mdt khoang thdi gian dai (Gandolfi et ai. 1989b).
Cac nghien ciru lai Ireland cho thay, mdi Iruong TC199 la tdt hon mdi Irudng Ham'F-lO khi nudi phdl ciing vdi te bao bieu md dng dan trirng (Lu et ai. 1987). Khi sii dung huyel thanh bd dgng duc (Oestrus Cow Serum) se thu dugc ket qua lot ban so vdl huyll thanh be bao thai (Fetal Calf Serum) {Wangetai, 1989).
Tai Phap, Marquanl-Le Guienne va ddng lac gid (1989) da tiln hanh nudi phdi bd trong mdi irudng B2 c6 bd sung OCS tren ldp te bao dng ddn irirng. Ngoai ra, co the sir dung mgt sd loai mdi trudng khac d l nuoi phdi nhu moi truong C R l a a , KSOM, SOF (Synthetic Oviduclal Fluid) (Dhali et
al, 2009 , Neira el ai, 2010 ; Leivas ei ai. 2011, V a r g a s / a / . , 2011).
KET L U A N
Te bao bieu md dng dan Ining cd anh hudng duong tinh den su phat trien ciia phdi bd thu tinh trong dng nghiem Ti le phdi dau-phdi nang d Id thi nghiem cd bd sung le bao dng dan trung la cao hon CO y nghia thdng ke {P<0,01) so vdi Id thi nghiem khong CO bd sung te bao, lan lugt 30,34% so vdi 17,20%
Ldi cam on. Nghien cim ndy nhdn duac su tdi tra tir de tdi cdp Bg Gido duc vu Ddo tao "Ndng cao hieu qud tao phoi bo thu tinh ong nghiim bang k\ thudt nuoi cdy chung v&i li bdo ong ddn tnrng "(Md so B20U-11-01J vd di ldi cdp Nhd nu&c "Uiig dung cong nghi cdy chuyen phoi bo sua cao sdn lai Tay Nguyen" (Ma sd TN3/C02).
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I N F L U E N C E O F B O V I N E O V I D U C T E P I T H E L I A L C E L L S O N T H E IN VITRO D E V E L O P M E N T O F IN VITRO F E R T I L I Z E D B O V I N E E M B R Y O S
Nguyen Huu Due'" *, Tran Thi Binh Nguyen', Le Thi Chau' Hanoi University of Agriculture
Tay Nguyen Institute for Scientific Research, Vietnam Academy afScienc e and Technology
SUMMARY
This study was conducted lo evaluate the effeci of Bovine Oviduct EpUhelial Cells (BOECs) on the in vilro developmeni of m vitro fertilized bovme embryos BOECs were isolated by modified method of Walter (1995). They were in vuro cultured by modified method of Kim el a! (2011). the m vitro culture was carried out in Sanyo incubator at 37''C, 5% COi, humidified air BOECs were cultured in supplemented Dulbecco's Modified Eagle Medium (D"MEM) BOECs' monolayer was developed in modified method of Mc Gowan (2011) Bovine oocytes were matured by modified method of Duc ef a/ (2003) Selected sperms were prepared by Swim-up method (modified method of Parrish et al. (1986h)), that was performed in Brackeu-Oliphant (BO) medium After the different penods (20, 30, 40, 50, 60, 70, 80 minutes), sperm concentration was automatically determinated by using Spermacue machine. In vUro fertilization belween the matured oocytes and selected sperms was conducted m Sanyo incubator ai at 5%C02. 39"C, humidified air (modified method of Duc €(fl/. (2003)) /H I'Uro fertilization ^^as earned out in BO medium supplemented 1 OfiM Fpinephenne. 10 jiM Hypotaurine, 20 pM Penicillamine, 10 pg/ml Hepanne. Bovine embryos were cultured in TC 199 medium supplemented Fetal Bovine Sreum 10%, in the presence or absence of the BOECs Results indicaicd that 1 After 60 minutes of Swim-up, we have got enough selected sperms' quantity for in vitro fertilisation. 2 Proportion of embryo at 2-4 cell and 4-8 cell stage in medium were noi significant differences (P-(l,n5i for non-BOCEs and BOECs tnals. that were 69,39% vs 70,34% and 54,71% vs 53,61%, rcspeaivciv >
' Aulhor for correspondence- E-mail: [email protected] vn
Nguyen HCiii Due et al.
Proportion of embryos at morula and blastocyst stages were significant differences (P<0,01), for non-BOCEs and BOECs tnals, thai were 17,20% vs. 30,34%, respecUvely. In conclusion, BOECs have affected positively the in vitro development of in vitro fertilized bovine embryos.
Keywords: BOECs, culture, embryo, fertdizanon. m vitro, maturation, oocyte, sperm
