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TAO PHOI BO
B A N GPHlTONG PHAP VI TIEM TINH TRUNG VAO BAO TirONG TRlTNG
Tran Thi Thanh Khu-ong, Dang Hodng Lam, Pham Van Phiic, Phan Kim Ngoc DH Khoa hoc Tu nhien TPHCM
MdDAU
De ndng cao hieu qud thy tinh nhdm nhdn nhanh ddn bd siia, viec dp dung vi tiem (microinjection) tinh trimg vdo bao tuong trirng la cdn thilt. Triing bd thu nhan tir nhieu nguon dugc nudi trudng thdnh trong TCM 199 vdi 20% FBS (Fetal Bovine Serum). Tinh triing bd sira ddng lanh dugc boat hod bdng phuong phap swim-up trong mdi trudng BO. Sau khi vi tiem, triing dugc boat hod. Sau 9 Idn thyc hien vdi 44 tning tao, dugc 20 phdi, vdi ty le thanh cdng Id 47,17% vd phdi dat giai doan morula la 10%.
ICSI - Intracytoplasmic sperm injection Id phuong phdp vi tiem mdt tinh triing dugc lya chgn tir trudc do vdo bdo tuong tning, vdi sy hd trg ciia he thdng vi thao tac ciing vdi kinh hien vi dao ngugc. Ky thuat ndy ra ddi vdo ndm 1992 do Palemo vd cs da ling dyng thdnh cdng tren ngudi. ICSI nhu Id mdt cudc each mang trong ho trg sinh san ddc biet trong chira tri vd sinh nam.
Ddi vdi gia siic, sau khi thyc hien ICSI, te bao tning cd chiia tinh triing phdi trai qua qud trinh boat hod vdi cdc tac nhdn nhu xung dien, ethanol 7%, Calcium lonophore (A23I87)...
I Tao phdi bd bdng phuong phdp vi tiem da md ra hudng mdi trong nhdn giong bd dem lai hieu qud cao, ddc biet Id bd sira trong trudng hgp ngudn giao tir cd chdt lugng kem hodc ngudn trimg ddng lanh. Ben canh dd, viec tiep can ky thuat hien dai cdn giiip hodn thien cdc quy trinh cdng nghe khdc trong cdc nghien ciiu xa nhu tao ddng, sinh thilt, tao ddng vat chuyen gen, bdo ton ddng vdt quy hiem...
V^T LI|:U VA PHU'ONG PHAP
Chuan bi tning: Trirng bd tuoi thu nhdn tai Id md hay tning ddng lanh dugc lya chgn, phdn loai A, B. Cdc trirng ndy dugc nudi trudng thdnh trong mdi trudng TCM 199 (Sigma) vdi 20% FBS (In vitrogen). Sau 24 gid nudi, tiln hdnh phd Idp tl bao cumulus bao quanh tning bdng enzyme hyaluronidase (Img/ml, Sigma).
338 HQI nqhj KHOA HQC VA C Q N G NGHg 2007
Hinh 1. Tru'ng bo chin.
(a) Trudc khi phd lap cumulus, (b) Sau khi pha Idp cumulus ". iG j Chuan bi tinh triing: Tinh trimg ddng lanh trong cgng ra dugc gidi ddng d 37°C trong 60 giay. Thyc hien swim-up 15 phiit trong mdi trudng BO, thu nhan dich ndi, chgn tinh trimg cho vi tiem.
Hinh 2. Vi tiem tinh trung vdo bdo tu'cypg tru'ng
Vi tiem: Hut tinh trimg bdng kim tiem (injection pipette) trong gigt PVP vitrogen) chuyen qua gigt TCM 199 chira trimg. Kim giir (holding pipette) trimg di dieu chinh the eye d vi tri 6 gid hay 12 gid, tiln hanh tiem tinh trimg vdo bdo tu(
trimg mdt each nhe nhang, nhanh chdng vdi thi tich dua vdo bdo tuong trimg it nhat
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339 Chuyen tning sau khi vi tiem vao mdi trudng nudi phdi CRlaa. Sau 4 gid nudi, tiln banh boat hod triing sau khi vi tiem vdi TCM 199 vdi 7% ethanol h-ong 4 phiit. Tilp tuc nudi phdi trong mdi trudng CRlaa vd theo ddi cdc giai doan phdt triln ciia phdi.
KET QUA VA THAO LUAN
• Sau 9 Idn vi tiem, dugc 44 hiing, sd phdi thu dugc la 20 phdi, chilm ty le tao phdi la 47,17%. Trong 20 phdi cd 2 phdi d giai doan 2 tl bdo (10%), 5 phdi giai doan 4 tl bdo (25%), 10 phdi giai doan 8 tl bdo (50%), 1 phdi giai doan 16 tl bdo (5 %) va 2 phdi giai doan morula (10%)).
(df.
Hinh 3. K i t qua tgo phoi bd bdng ICSI.
(a) Phoi 2 tl bao, (b) Phoi 4 tl bao, (c) Phoi 8 tl bao, (d) Phoi morula
Ndm 1989, Younis vd cs da tao dugc phdi bd bdng ky thu^t ICSI truyin thdng vd tning khdng boat hda vdi ty le 2%. Li vd cs (1999) tao phdi bdng piezo-ICSI cd hoat hda bdng ethanol 7% phdi tao ra vdi ty le 26,4%. Cimg ndm do Hamano dd thdnh cdng vdi ty le tuong doi cao la 56,6 %. Ndm 2004, Kato, H., Matsumoto vd cs da so sdnh giua boat hda bdng ethanol 7% vd khdng boat hda, dat ty le phdi Id 51% so vdi khdng hoat hda Id 13%. Takenaka vd cs (2005) cimg dimg phuomg phdp boat hda bang ethanol 7% vdi ty le tao phdi la 75,6%.
340 HQi nghi KHOA HQC VA C O N G NGHE 2007
Nhu vay vdi ty le tao phdi la 47,17% la tuong ddi cao, cao hon hdn vdi lan thyc hien dau tiln vao nam 1989. So vdi cdc nghien ciru gan day thi ty le tao phdi la tuong ddi thdp, tuy nhien cdc nha khoa hgc tren thi gidi thyc hien ICSI cd ho trg Piezo frong khi chiing tdi thyc hien vdi phuong phap cd diln, dimg lyc co hgc cua kim tiem vao mdng tning. Ky thuat Piezo-ICSI, mdng pellucida va mang noan se chiu it sy tdc ddng ciia ddu pipette ma thay vao do la cd sy ho trg ciia tia piezo. Mdt trong nhiing thuan Igi cua ky thuat piezo la gidm dang kl sy pha buy tning hong sudt qua trinh thy tinh. Trong khi vdi ky thuat cd diln, lyc ciia ddu kim gdm lyc tilp tuyIn vd lyc xoay da phdn nao tac ddng din tl bdo chdt tning.
Nhu vay, vdi ky thuat ICSI cd diln chimg tdi da tao ra phdi vdi ty le 47,17% cimg vdi sy boat hda ciia ethanol 7%. Tuy ethano 17% khdng phdi la tdc nhdn boat hda nhan tao tdi uu nhung ty le tao phdi ciing tuong ddi cao.
Cd the khang dinh rang vdi ngudn mau dn dinh, trimg chat lugng tdt, he thdng vi thao tdc tdt, dn dinh cimg vdi tay nghe cua ky thuat vien thao tdc cao thi ty le phdi bo bang phuomg phdp ICSI khdng chi dimg d ty le 47,17% md cdn cao hon nira.
Ddi vdi tning ngudi, ty le ICSI cao hon so vdi IVF, nhung d bd: theo Kim tea Chung vd cs (1999) ty le phdi IVF Id 75%, cua Phan Kim Nggc vd cs (2006) Id 72,19%
ty le tao phdi bdng ICSI thdp hon do bdn chdt ciia tning bd khd boat hda hon so vdi hiing ngudi va dk tao thdnh hgp hi, chung phdi nhan nhimg kich thich nhdn tao. Tuy nhien, Gyu-Jin Rho va cs (1998) da tiln hanh xac dinh gidi tinh tinh trimg bdng phuong phap Flow Cytometry vdi ty le 80% vd tiln hanh vi tiem tinh trimg da xdc dinh gidi tinh. Ty le phdi dat dugc vdi phuong phdp nay Id 46,6% vd phdi d giai doan blastocyst la 6,9%. So vdi kit qud tao phdi ciia chimg tdi Id 47,17 % cao hon. :: i , |
KET LUAN 5 Trong phuong phap chudn bi giao tir, swim-up dimg cho boat hod tinh trimg phii
hgp vdi ICSI. Tac nhan ethanol 7% cd vai frd hoat hod tning sau khi vi tiem, cho kit qua tao phdi tuong doi cao va phii hgp vdi dilu kien thyc hien. Bdng ky thuat ICSI co dien, vdi tac nhan hoat hod la ethanol 7% ty le tao phdi 47,17% vdi phdi dat giai doan morula la 10%.
TAI LIEU THAM KHAO
1.
i
Carlos A. Guerrero. (2006) Cryopreservation And Intracytoplasmic Sperm Injection With Bovine Epididymal Spermatozoa. .S. Louisiana State University.
Henry E. Malter 8c Jacques Cohen.(2002) Intracytoplasmic sperm injection:-,
technical aspects. Gamete source, manipulation and disposition. d Heuwieser W, Yang X, Jiang S, Foote RH: Fertilization of 21. Yanagida t},
Katayose H, Yazawa H, et al(\992): Successful fertilizabovine oocytes qfiefj microsurgical injection of spermatozoa Theriogenology
Ph^n IV: CdNG NGHg SINH HQC B Q N G VAT 3 4 j
4. Jin-Tae Chung (1999). Activation of bovine oocytes following intracytoplasmic sperm injection (ICSI). Department of Animal Science Macdonald Campus McGill i University, Monfreal, Quebec, Canada.
5. Keith J. Betteridge (2004). New reproductive Technologies in Cattle: A Veterinary Perspective. Department of Biomedical Sciences, Ontario Veterinary College, f. University of Guelph, Guelph, Ontario, NIG 2W1, Canada.
6. Kimura, Y & R. Yanagimachi (1995/ Intracytoplasmic sperm injection in the mouse. Biol. Reproduction. 52: 709-720.
7. Mapletoft RJ & J. F. Hasler (2005). Assisted reproductive technologies in cattle: a review. (1) Westem College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada (2) AB Technology, Bioniche Animal Health USA, hic, Pullman, WA 99163, United States of America.
8. Ragaa Mansour (1998). Intracytoplasmic sperm injection: a state of the art technique Human Reproduction Update, Vol. 4, No. 1 pp. 43-56.
9. Toshitaka Horiuchi (2002) Application of Intracytoplasmic Sperm Injection to Animal Production. Faculty of Life and Environmental Sciences Department of Life Sciences.
SUMMARY
Bovine embryo production by intracytoplasmic sperm injection
Tran Thi Thanh Khuong, Dang Hoang Lam, Pham Van Phuc, Phan Kim Ngoc University of Sciences HCMCity
Due to bovine oocyte source is rare. To increase fertilization effect, application of infracytoplasmic sperm injection method is nessesary. In our research, bovine oocytes are collected at slaughter-house and cultured with TCM 199 plus 20% FBS (fetal bovine semm) until maturation. Cryopreserved bovine semen is thawed and activated with BO medium. After injection, oocytes are freated with 7% ethanol in TCM 199 for 4 min for activation. In 9 repeats, 44 oocytes were injected, with 20 embryo that are formed, with 47,17% cleavage rate, 10% morula embryo.