PHN IV

CNG NGH SINH HC NG VT

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THU NHN V NUI CY T BO SINH DNG BO GM Neofelis nebulosa

Hong Ngha Sn, Trn Cm T, Vin Sinh hc Nhit i L Vn Ty, Vin Cng ngh Sinh hc

M U Bo gm Neofelis nebulosa(Griffth, 1821), h mo Felidae, b n tht Carnivora, thuc c ln trong h mo, sng rng rm nhiu tng trn ni t, ni [1]. Vit Nam bo gm phn b Tuyn Quang, Lai Chu, Lng Sn, Sn La, Vnh Ph, Ho Bnh, Kon Tum, Gia Lai, Lm ng, ng Nai, y l loi th qu him, cho da lng v dc liu. Hin nay bo gm tr nn qu him, trong Sch Vit Nam (2000), bo gm c xp cp V (s nguy cp) [1]. CITES, Hip c v bun bn quc t cc loi ng, thc vt hoang d ang gp nguy him, a bo gm vo cc loi ca ph lc I, cm bun bn [2]. Nc M cng a bo gm vo trong Chng th cc loi ang gp nguy him, nhm ngn chn vic bun bn bo gm hay cc b phn c th chng. Vi nhng tin b vt bc ca khoa hc trn th gii v k thut bo tn ngun gen cp t bo, k thut cy nhn to phi clonning... cng nhng ch trng, chnh sch ca ng v Nh nc Vit Nam v bo v cc loi ng vt hoang d th vic tin hnh thu nhn, nui cy t bo sinh dng bo gm nhm bo tn ngun gen loi ny cp t bo l rt cn thit.Vic bo v ngun gen cp t b o s gip chng ta gim bt chi ph cho cng vic bo tn ng thi phng php ny cng d dng thc hin v thch ng cc loi iu kin, giai on thu thp mu t bo soma n gin v d thc hin, ngay c vi nhng ng vt mi b cht. Tuy nhin ti to ra nhng con vt thc s theo ph ng php nhn bn th i hi phi c nhiu thi gian v tin bc.

Hnh bo gm nui nht ti Tho Cm Vi n

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Kt qu bc u thu nhn c: nguyn bo si ca bo gm pht trin c trong mi trng DMEM b sung 10 - 15% FBS. i hi v nng FBS ca t bo bo gm l cao hn so vi t bo b rng, b tt. Kh nng bm dnh v lan to t bo ca mu ti tt hn mu ng lnh. VT LIU V PHNG PHP Ha cht: Dung dch PBS(-); Cn 70o (China); Mi trng DMEM (Gibco); FBS (Gibco); Trypsin (Sigma); EDTA (Merk); Non -essential amino acid (Sigma) Vt liu: Mnh da tai bo gm c thu nhn ti Tho Cm Vin Thnh ph H Ch Minh.
Phng php

Phng php nghin cu: Nghin cu theo phng php thc nghim m t. Phng php thu nhn v x l, ng lnh mu: Mu da tai bo gm sau khi c thu nhn c ra sch trong mi trng PBS (-); mt na cho vo tp ng lnh cha 1ml PBS 10% DMSO ng lnh v bo qun trong ni t lng -196oC (ng lnh chm). Na cn li cho vo effendorf chuyn v phng th nghim T bo ng vt - Vin Sinh hc Nhit i. Mu tai b rng c x l qua nhng bc sau: effendorf cha mu c xt cn 70 0C ri a vo t v trng. Mu c ra qua dung dch PBS (-) 3 ln, ri tin hnh co sch lng. Sau , ngm mu v o cn trong vng 30 giy v ra li 3 ln bng dung dch PBS( -). Phng php nui s cp mu m: Tin hnh ct nh mu m thnh nhng mnh c kch thc 0,5x0,5mm. Sau , gp nhng mnh m v o a nha 4 ging. Mi ging t 4 n 5 mnh m. Ch t 20 n 30 pht cho mu m c nh tr n mt a. Sau khi mu c nh b sung 400l mi tr ng DMEM 10%FBS v thm 1% non-essential amino acid. Chuyn vo t m 37-38oC, 5%CO 2 nui cy. Sau mi 48 gi kim tra v ghi nhn hnh nh. Phng php cy chuyn: Gp b mnh m v b sung mi trng sau 5 - 7 ngy nui, quan st s pht trin ca nguyn bo si, khi thy t bo lan ht mt a th tin hnh cy chuyn.

Hnh 1: Chun b dng c th nghim

Hnh 2: Mu m tai bo gm

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Ht b ht mi trng c ra, b sung vo 200l trypsin/EDTA 0,25%, l c nh a t bo tch khi mt a. Quan st di knh hin vi, khi thy t b o bung ra ht th b sung thm 200l mi trng DMEM 10%FBS bt hot trypsin. Ht 200l huyn ph t bo vo a 4 ging mi v b sung thm 200l mi trng DMEM 1015%FBS. Tip tc nui trong t m 37-38oC, 5%CO2 v theo di kt qu pht trin. KT QU V THO LUN
1. Kt qu nui cy s cp

Khi c nui nguyn pht t mnh m, sau 7 ngy nui t bo mc lan ra v sau 14 ngy t bo lan ht b mt a 4 ging, mu m c gp b ra khi mi trng nui cy v c th tin hnh cy chuyn ra a 4 ging mi.
2. Kt qu sau khi cy chuyn ln 1

Sau khi c cy chuyn ln 1, cc t b o tip tc phn chia v pht trin mnh trong mi trng nui cy. Sau 10 ngy t bo mc lan ht b mt a 4 ging v t mt cy chuyn qua a mi. Khi nui cy in vitro, cc t bo bm trn b mt a a phn c hnh thoi thon di, c nhn to hnh cu. Tip tc cy chuyn khi t b o mc lan trn 80% din tch ca a nui. T bo thu c sau trn 2 ln cy chuyn l nguyn bo si c hnh dng l hnh sao hoc hnh thoi, c nhn to hnh cu.

Hnh 3: T bo lan to sau 7 ngy nui s cp Hnh 4: T bo lan to sau 10 ngy nui

Hnh 5: t bo sau 7 ngy cy chuyn ln 1 Hnh 6: T bo sau 10 ngy cy chuyn ln 1

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Nh vy, qun th nguyn bo si thu c chng t nui cy c t bo sinh dng ca bo gm trong mi tr ng DMEM 10% FBS, 1% non -essential amino acid thu nhn s lng ln t bo nhm bo tn ngun gen cp t b o, to nguyn liu cho nhng nghin cu su hn nh chuyn nhn, to dng v tnh Tip tc th nghim thm nhiu nng huyt thanh khc kho st nng huyt thanh ti u cho s pht trin t bo bo gm, chng ti nhn thy tc pht trin v lan to ca t bo bo gm trong mi trng c b sung 15% FBS nhanh hn so vi ch b sung 10% (qua theo di c nh gi mc +++ 10% FBS v ++++ 15% FBS).
2.3. So snh tc pht trin ca t bo mu ti v ng lnh

Mu m ng lnh trong nit lng -196oC c gii ng bng phng php gii ng nhanh, cho mu vo ngay b nc m 37oC trong thi gian 30 giy n 1 pht sau tin hnh st trng v thc hin cc thao tc khc nh mu ti. Nhn chung, cng nh kt qu nui cy mu m ca b rng, b tt th kh nng bm dnh v tc lan to t bo ca mu m bo gm ti tt hn so vi mu ng lnh. i vi mu ti th ngy nui th 3 mu m bm dnh vo y ging nui, t l mu bm dnh l khong 80 - 85% v ngy th 5-6 quan st thy t bo lan to ra xung quanh. i vi mu ng lnh th ngy th 3-4 mu m bt u bm vo y ging nui nhng mc bm dnh cn cha tt, t l cc mu bm dnh cng ch 60 70%; quan st thy t bo bt u lan to ngy 6-7.
Ch tiu theo di Kh nng bm dnh Tc lan to Mu ti Ngy 3 +++ Ngy 5-6 +++ Mu ng lnh Ngy 3-4 ++ Ngy 6-7 +++

TI LIU THAM KHO 1. 2. 3. B Khoa hc, Cng ngh v Mi trng (2000). Sch Vit Nam. Phn I: ng vt. NXB KH&KT IUCN, 2004: Red List of threatened species. Chnh ph nc CHXHCN Vit Nam, 2002: Ngh nh 48/2002/N-CP v sa i, b sung danh mc thc vt, ng vt hoang d qu him ban hnh theo N 18/HBT ngy 17/01/2002 c a Hi ng B trng. L Vn Ty v cs, 2003: Bc u to phi nhn bn b tt (Bos Gaurus) bng k thut cy nhn lm c s cho vic bo v loi ng vt qu him ny ca Vit Nam. Tp ch Sinh hc 25(2): 1-6. Fresney, I. R (1984). Culture of animal cells: A manual of basic tecnique . Aln R. Liss, Inc., New York. Frederick M. A (2003). Current protocol in Molecular biology. Mammalian cell culture. Chapter 28

4.

5. 6.

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7.

Hong Ngha Sn v cs, 2007: Thu nh n v nui cy t bo sinh dng ca b rng Bos javanicus (Wag rer, 1844) nhm bo tn ngun gen cp t bo. Tp ch Sinh hc.

SUMMARY

First results of collect and culture of Neofelis nebulosa somatic cells

Hoang Nghia Son, Tran Cam Tu, Le Van ty Institute of tropical Biology

The Neofelis nebulosa tissue was collected at Thao Cam Vien Zoo Park (Ho Chi Minh city, Vietnam) and cultured at Institute of Tropical Biologys laboratory. According to the results of experiment , Neofelis nebulosa cells can growth we ll in the DMEM medium added 10 - 15% FBS and 1% non-essential amino acid. The growth rate of fresh tissue is better than the freezing tissue. Large number of typical firoblasts was collected and conservation in nitrog en (-196oC) for the production of embryos of an endangered species (Neofelis nebulosa) in the future.

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THU NHN V NUI CY T BO SINH DNG CA B RNG Bos javanicus NHM BO TN NGUN GIEN CP T BO
Hong Ngha Sn, Trn Cm T, Vin Sinh hc Nhit i L Vn Ty, Vin Cng ngh Sinh hc

M U B rng Bos javanicus (Wagrer, 1844) thuc h Bovidae, l loi th c ln, c trng lng c th 600 - 800kg. B rng thch sng nhng v ng rng tha thong mt, nht l rng khp hoc nhng khu rng rm c thung lng c nhiu c. Vit Nam, trc y b rng c nhiu cc tnh ng N ai (Bin Ho, La Ng), B Ra - Vng Tu, Bnh Thun (Phan R - Phan Thit), Lm ng, c Lc y l loi th qu him ca rng nhit i, th ng b sn bn nhiu ni nn ang ng trc nguy c tuyt chng. Trong Sch Vit Nam (2000), b rng c xp cp V (s nguy cp) [1]; trong Danh lc ca IUCN (2004), b rng mc EN (nguy cp) [2]; cn trong Ngh nh 48/2002/N-CP ca Chnh ph, b rng c xp trong Ph lc IB [3]. Do vy, vn bo tn ngun g en l mt trong nhng nhim v chin lc ca cng ngh sinh hc Vit Nam. Trn c s t bo sinh dng ca loi b rng c kh nng pht trin trong mi trng DMEM c b sung huyt thanh, ng thi da vo nhng iu kin sn c ca phng th nghim, chng ti tin hnh thu nhn v nui cy t bo sinh dng ca b rng nhm bo tn ngun gien cp t b o v lm nguyn liu to dng v tnh v sau ny. Kt qu thu c l t bo sinh dng ca b rng pht trin tt trong mi tr ng DMEM c b sung 10% huyt thanh ca b o thai b v thu c s lng ln t bo fibroblast ca b rng. VT LIU V PHNG PHP Ha cht: Dung dch PBS(-); cn 70o (China); mi trng DMEM (Gibco); FBS(Gibco); trypsin (Sigma); EDTA (Merk); Polyvinyl alcohol (China); L glutamine (Sigma); D-glucose (Sigma) Vt liu: Mu m ca tai b rng c thu nhn ti c s nui th hoang d khu vc Tuyn Lm, thnh ph Lt. Phng php: Nghin cu thc nghim c tin hnh ti Vin Sinh hc Nhit i v Trng i hc Quc gia Thnh ph H Ch Minh.

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Phng php thu nh n v x l mu: Mu tai b rng sau khi c thu nhn, c ra sch trong mi tr ng PBS(-); mt na cho vo tp ng lnh cha 1ml PBS 10% DMSO ng lnh. Na cn li cho vo effendorf chuyn v phng Th nghim t bo ng vt, Vin Sinh hc Nhit i. Mu tai b rng c x l qua nhng b c sau: effendorf cha mu c xt cn 70 o ri a vo t v trng. Mu c ra qua dung dch PBS ( -) 3 ln, ri tin hnh co sch lng. Sau , ngm mu v o cn trong vng 10 giy v ra li 3 ln bng dung dch PBS(-). Phng php nui s c p mu m: Tin hnh ct nh mu m th nh nhng mnh c kch thc 0,5x0,5mm. Sau , gp nhng mnh m v o a nha 4 ging. Mi ging t 4 n 5 mnh m. Ch t 20 n 30 pht mu m c nh trn mt a. Sau khi mu c nh, b sung 400l mi tr ng DMEM 10%FBS v chuyn vo t m 37 - 38 oC; 5%CO 2 nui cy. Sau mi 24 gi, kim tra v ghi nhn hnh nh. Phng php cy chuyn: Mu m c nui sau 5 ngy, gp b mnh m v b sung mi trng. Quan st s pht trin ca nguy n bo si, khi thy t bo lan ht mt a th tin hnh cy chuyn. Ht b ht mi tr ng c ra, b sung vo 200l trypsin/EDTA 0,25%; l c nh a t bo tch khi mt a; quan st di knh hin vi, khi thy t b o bung ra ht th b sung thm 200l mi trng DMEM 10%FBS bt hot trypsin. Ht 200l huyn ph t bo vo a 4 ging mi v b sung thm 200l mi tr ng DMEM 10%FBS. Ti p tc nui trong t m 5%CO 2. Sau mi 24 gi, kim tra v ghi nhn hnh nh. KT QU V THO LUN
1. Kt qu nui cy s cp mu m da tai b rng

Khi c nui nguyn pht t mnh m, cc nguyn bo si tng sinh rt nhanh, di c ra khi mu m ban u v pht trin rt tt sau khi gp b mnh m ra khi mi tr ng nui cy. C th quan st c rt nhiu t bo phn chia trong a nui vi nhiu hnh dng khc nhau; sau 4 ngy, t bo mc lan ra v sau 10 ngy, t bo lan ht b mt a 4 ging v c th tin hnh cy chuyn ra a 4 ging mi.

Hnh 1. Sau 4 ngy nui s cp

Hnh 2. Sau 10 ngy nui

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Hnh 3. T bo sau 10 ngy nui (X20)

Hnh 4. T bo sau 10 ngy nui (X10)

2. Kt qu sau khi cy chuyn ln 1

Sau khi c cy chuyn ln 1, cc t bo tip tc phn chia v pht trin mnh trong mi trng nui cy. Sau 6 ngy, t bo mc lan ht b mt ca a 4 ging v t mt cy chuyn qua a mi. Khi nui cy in vitro, cc t bo bm trn b mt ca a a phn c h nh sao hoc hnh thoi, c nhn to hnh cu. Tip tc cy chuyn khi t b o mc lan ra hn 80% din tch ca a nui.

Hnh 5. Sau khi tch b ng trypsin/edta 0,25%

Hnh 6. T bo sau 24 gi c cy chuyn ln 1

Hnh 7. T bo sau 48 gi cy chuyn ln 1

3. Kt qu sau khi cy chuyn ln 2

Sau khi cy chuyn ln 2, nguyn bo si vn tip tc phn chia v lan ht b mt ca a nui sau 6 ngy. Hnh dng ca t bo c hnh sao hoc thoi, c nhn to hnh

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cu. iu ny chng t, sau khi c nui cy trong mi trng DMEM 10%FBS v cy chuyn nhiu ln, t b o thu c l nguyn bo si.

Hnh 8. T bo sau 48 gi c cy chuyn ln 2 (X20)

Mc tiu t ra l nui cy thnh cng nguyn bo si t mu tai b rng. Trong qun th t bo thu c sau khi cy chuyn 5 ln: - Ch yu l nguyn bo si in hnh: c hnh thoi, hnh sao - C s sinh sn vi phn bo in hnh Nh vy, qun th nguyn bo si thu c chng t nui cy thnh cng t bo sinh dng ca b rng trong mi trng DMEM 10%FBS thu nhn s lng ln t bo nhm bo tn ngun gen cp t bo, to nguyn liu cho nhng nghin cu su hn nh chuyn nhn, to dng v tnh

Hnh 9. T bo sau 48 gi c cy chuyn ln 2 (X10)

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TI LIU THAM KHO 1. 2. 3. B Khoa hc, Cng ngh v Mi trng (2000). Sch Vit Nam. Phn I: ng vt. NXB KH&KT IUCN, 2004: Red List of Threatened Species. Chnh ph nc CHXHCN Vit Nam (2002). Ngh nh 48/2002/N-CP v sa i, b sung danh mc thc vt, ng vt hoang d qu him ban hnh km theo N 18/HBT ngy 17/1/2002 ca Hi ng B trng. Fresney, I. R, 1984: Culture of animal cells: A manual of basic technique . Aln R. Liss, Inc., New York. Nguyn Phan Xun L (2006). Thit lp quy tr nh nui nguyn bo si t da quy u ngi. Lun vn Thc s Sinh hc. Tr ng H Khoa hc T nhi n TP. HCM. L Vn Ty v cs, 2003: Bc u to phi nhn bn b tt (Bos Gaurus) bng k thut cy nhn lm c s cho vic bo v loi ng vt qu him ny ca Vit Nam. Tp ch Cng ngh Sinh hc 25(2):1-6 L Vn Ty v cs, 2003: Nhn nui, bo qun ng lnh t bo, to phi bng k thut cy nhn nhm bo v a dng sinh hc lo i b tt. Hi ngh Cng ngh sinh hc ton quc: 712 - 716, Nxb KH&KT

4. 5. 6.

7.

SUMMARY

Collect and culture of bull somatic cell of Bos javanicus for gene conservation
Hoang Nghia Son, Tran Cam Tu(1), Le Van Ty(2) (1)Institute of Tropical Biology (2)Institute of Biotechnology

Vietnam is a tropical country which has plentiful animals and plants, especially wide animals. Many animal species were listed in t he Red Data Book of Vietnam (2000). However, many animal species were illegally hunted for aesthetic and economic purposes, for example bull. According to some points of view, bull somatic cells had the ability to develop in the DMEM added serum; besides, in our lab conditions, we have collected and cultured bull somatic cells to gene conservation for the making of materials for cloning later. Study results showed that bull somatic cells grew well in the DMEM medium added 10% FBS and we have collected a lar ge number of typical firoblasts.

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CI TIN V THCH NGH CC K THUT GY NG DC V RNG TRNG TRN B SA H LAN NHM GII QUYT VN CHM SINH V RT NGN KHONG CCH GIA HAI LN SINH CON
L Van Ty, Vin Cng ngh Sinh hc Hong Ngha Sn, Vin Sinh hc Nhit i Nguyn Mng Hng, H KHTN, H Ni

M U Cc nghin cu c bn v bung trng ng vt c v trong nhng nm gn y chng minh quy lut pht trin qun th nang bung trng khng tnh tin m theo dng sng. Bt u t mt s nang c kch th c ti hn, di nh hng ca cc gonadotropin cc nang ny tham gia vo m t qu trnh pht trin, cnh tranh nhau dn n phn ho v tc ln. Ch c cc nang c tc pht trin nhanh n c giai on c tn nang. Cc nang pht trin chm h n trc sau u b ri vo trng thi thoi ho, cht i v tiu bin vo lp ni mc bung trng. Sng nang mi ch c th hnh thnh khi nang c tn pht trin n giai on rng trng hoc b thoi ho. Trng rng tt c cc sng nang c chng minh l u c kh nng th tinh, to phi tng t so vi trng rng cui chu k [3, 7]. V l thuyt, iu n y m rng kh nng p dng ca cc k thut sinh sn: trng thi sinh l bung trng c xem l lp li sau mi sng nang ch khng ch mt ln trong mt chu k nh quan nim trc y. b sa, ngi ta quan st thy khong 70% s c th c chu k 2 sng nang, 30% s c th c chu k 3 sng nang [1, 2]. Nh vy c hi p dng cc k thut sinh sn nh gy ng dc v rng trng, gy siu bi non, ly trng bng chc non (ovumpckup),... i vi b tng ln 2 n 3 ln. Trong th tinh nhn to b, gy ng dc v rng trng ng lot c vai tr quan trng. Thay v cho vic quan st ng dc hng ngy, tri di sut trong nm tn nhiu nhn cng m ch t c kt qu rt dao ng, gy ng dc ng lot to iu kin cho tp trung theo di ng vt trong thi gian ngn, dn tinh ng thi im cn thit, chng nhng nng cao hiu qu th tinh m cn c th ch ng cho b cha vo thi gian mong mun. i vi b sa, thi im dn tinh tr li c tnh ton kinh t nht l 60 n 120 ngy sau khi b [1]. Khong cch ny cng ko di, cng ko di kho ng cch gia hai la gy thit hi v kinh t (sn l ng sa, s b con sinh ra) cng ln. i vi n b cao sn th thit hi ny cng r nt.

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Nui b sa hin nay nc ta ang tr thnh mt ngh sn xut hng ho. Nng sut sa, s b con sinh ra l nhng ch tiu quan trng quyt nh mc thu nhp v i sng ca ngi chn nui. Ngoi ra do bin ng ln v thi tit, v thc n theo ma v chn thi im sinh b ca n b ni chung v n b sa ni ring cng c ngha rt quan trng. Ch ng cho b sinh con vo lc th i tit thun li, lc di do thc n, b m cho sa nhiu hn, b con c t l c nui sng cao hn. Gii quyt cc vn nu trn mt mt tng thu nhp cho ng i chn nui, mt khc hn ch c hi chng bt dc m a v, vn v sinh cng nh s dng hiu qu thi gian c ch i vi mi b sa. Trong bi vit ny chng ti s a ra th nghim mt s quy tr nh gy ng dc v rng trng p dng c th tr n n b sa ca Sn La vi hy vng tm ra mt, hai quy trnh ti u p ng cho vng chn nui b sa ny cng nh m rng cho cc a phng khc. VT LIU V PHNG PHP ng vt: i tng nghin cu l n b sa H Lan, chn nui ti cc h gia nh cc i sn xut Sng L - Bc Quang, 3/2, 26/3, Bnh Minh (Nng trng T Hiu, huyn Mai Sn, tnh Sn La). y l n b sa nhp t Australia trong hai t 12.2002 v 06.2003 vi tng n vo thi im th nghim l 406 con. S b c chn ra tham gia v o th nghim l 145 con. Ton b s b th nghim c nui trong gia nh cc h chn nui. S b ci trong mi h c t 3 n 12 con b. B c nui dng vi ch dinh dng qui nh cho b sa thng nht ca Nng tr ng. Ch cc con c th trng tt (thang im > 5), khng bnh tt hoc iu tr c ch nh tham gia th nghim. Cc kch thch t: GnRH(Gonadotropin releasing hormone), PMSG (Folligon), HCG(Corulon), PG (Prosolvinl), Vin c y tai SMB (Crestar), [Intervet, H Lan]; Estradiol [Hanvet-Vit Nam]. M t quy trnh: B c kim tra khng b cc bnh sinh sn, khng c cc vn v dinh dng, th trng tt, khm qua trc tr ng kim tra thy c th vng mt trong hai bung trng c ch nh p dng th nghim cc quy tr nh ny. Qui trnh I: GnRH-PG-PMSG: Tin hnh tim c mng 2 ml ch a 10?g GnRH vo ngy khm coi l ngy th nht, by ngy sau, tim 1 mi PG 2 ml, cng lc tim 500 iu PMSG. Sau GnRH c th c mt s b ng dc, s ny c khm kim tra t l rng trng. Sau mi ti m PG v PMSG theo di ng dc v dn tinh 2 ln theo quy tc AM/PM. Quy trnh II: GnRH-PG-E: Tin hnh nh quy trnh I, thay th mi tim PMSG bng mi tim 5 mg estradiol benzoate

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Quy trnh III: PG-PG-HCG: Tin hnh nh quy trnh I, vi mi tim th nht GnRH c thay th bng 2ml PG v mi tim PG th hai kt hp vi mt mi tim HCG: 2.000 iu. Qui trnh IV: SMB-PG-PMSG: Thi im vin cy tai Synchno-mate B (SMB) c cy vo gc tai b kt hp vi tim c 5 mg estradiol valerate c tnh l ngy th nht. 6 ngy sau, tim 2ml PG kt hp vi 500 iu PMSG, ng y th 7 tho b vin cy tai. Theo di ng dc v dn tinh 2 ln theo quy tc AM/PM Qui trnh V: PG+Gn RH PG+GnRH (Ovsynch): Cc b ny c tim 2ml PG c ho sn vi 10g GnRH, ngy tim c tnh l ngy th nht. Sau 7 ngy tim lp li 2 ml PG v 10?g GnRH. Ghi nhn b ng dc sau mi tim th nht, kim tra t l rng trng bng khm qua t rc trng, theo di ng dc v dn tinh 2 ln theo quy tc AM/PM sau mi tim th hai. Th nghim rt ngn thi gian gia hai la : 69 b 1 thng tr ln vo thi im th nghim c kim tra b my sinh dc bng cch khm qua trc tr ng nu thy t cung phc hi, khng c du hiu bnh l, bung trng pht trin b nh thng c ch nh cho th nghim rt ngn thi gian gia hai ln . Quy trnh Ovsynch (quy trnh V, ph n trn) m rng p dng th nghim rt ngn thi gian gia hai ln . KT QU V THO LUN 1. Th nghim 5 quy trnh Tng hp cc kt qu kho st 5 quy tr nh (bng 1) cho thy: t l ng dc, t l u thai cao nht (100%) v tp trung nht quan st thy khi p dng vi vi n cy tai SMB kt hp vi PG v PMSG (quy trnh IV). y l quy trnh kt hp c tt c cc li th: liu estradiol ban u gy thoi ho cc th v ng mi hnh thnh; duy tr lin t c mc progesterone cao c ch b i tit LH, duy tr c tn nang, tim PMSG lm gia tng pht tri n nang. Trn nn nh vy, khi tim PG gy thoi ho th vng cng vi vic ly i vi n cy tai, lm gim t ngt mc progesterone trong mu gy ng dc v rng trng. ng dc rt tp trung (trong vng t 48 n 72 gi), ng pha gia ng dc v rng trng l rt cao, biu hin l 100% s b ng dc v c cha. Tuy nhin, y cng l mt quy trnh c gi thnh cao nht, x l phc tp nht. Quy tr nh gy ng dc nh th ny ch p dng i vi nhng b cao sn k lc hoc cho vic cy phi t i khi yu cu chnh xc thi im ng dc. Ci tin quy trnh Ovsynch (quy trnh V) so v i quy trnh nguyn gc [6] l thay v tim mi GnRH th 2 v o 48 gi sau PG, chng ti tim GnRH ng thi vi PG. ng dc ca b km tp trung hn, c khong 7% s b ng dc trong vng t 72 n 96 gi sau khi tim. Tuy nhin, nu khng dn tinh v thi im c nh nh trong quy trnh chun, m theo di ng dc v dn tinh theo

324

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quy tc AM/PM th vn t t l th thai cao (100% so vi s b ng dc, 70% so vi s b x l). Mt khc, do khng b it r trng thi nang vo thi im x l, mi tim u tin kt hp gia PG v GnRH va kch thch bi tit FSH, LH va lm thoi ho th vng (giai on sm do GnRH, giai on mun do PG), to iu kin ng dc v rng trng tp trung h n sau mi tim th 2. Quy trnh kinh in vi hai mi ti m PG cch nhau 7 ngy c h n ch l c t l b khng ng dc cao v khng tp trung [4]. Vic thay th mi PGF2 u tin bng GnRH kch thch b i tit FSH, LH, kch thch pht trin nang trng. Do vy mi PG th 2 gy thoi ho th vng ng vo lc nang pht tri n n giai on cn rng trng. Chn la PMSG (500 iu) va kch thch ln nang v a gy rng trng, estradiol (5 mg) kch thch ln nang hoc HCG (2000 iu) kch thch rng trng, hon ton ph thuc vo ngha kinh t ca mi quy tr nh. Tuy nhin trong cng trnh ny, ng dc tp trung h n quan st thy khi tim PG kt hp vi HCG (48 n 72 gi), nhiu b ng dc hn nu c tim PG kt hp vi estradiol (75%). Tim k t hp vi estradiol mc d cho t l ng dc cao h n nhng t l u thai li thp h n (50%) trong khi t l u thai b tim PG kt hp vi PMSG l tng ng vi b tim PG kt hp vi HCG (70,6% v 72,2%) C t l ng dc t ti 75% v t l u thai t 50% s b x l chng t quy trnh dng kt hp vi estradiol gy ng dc ng thi vi gy rng trng. Vn t ra l phi iu chnh liu l ng estradiol th n o hn ch hiu ng gy cht phi do tc ng gy co bp t cung ca hooc -mn ny. PMSG vi liu lng 500 iu/ b kch thch pht trin nang, khng gy tng sinh bung trng. Hiu ng LH ca hp cht n y kch thch rng trng. Tuy nhin do thi gian bn phn r ca PMSG ko di nn mc d c t l ng dc cao nhng ng dc khng tp trung m ko di t 24 n 120 gi sau khi tim thuc. Mt khc PMSG liu n y cng lm tng tn s rng trng kp v sinh i. Quy trnh tim kt hp vi HCG cho tp trung ng dc cao trong v ng t 48 n 72 gi v cho t l u thai cao (72,2%). Tuy nhi n do tim hai liu PG cch nhau 7 ngy nn c t i 41% s b khng ng dc, do vy thay th mi ti m u bng GnRH chc chn s nng cao hiu qu ca quy tr nh . 2. Kt qu th nghim rt ngn thi gian gia hai la Do cho t l u thai cao v ng dc tp trung, Ovsynch ( quy trnh V) c chng ti m rng p dng trn 65 b sa HF. Kt qu p dng quy tr nh rt ngn thi gian t lc b sinh con n lc b c cha li l: 91+ 28 ngy so v i vi ton n (c theo d i ng dc t nhin v dn 2 ln cho mi ln ng dc) l : 214 + 152 ngy (bng 2). p dng quy trnh V cho b vo thi im trn mt thng sau khi b cho s b u thai c th t 85,5% s b c cha sau 4 thng, a nh phn b t l u thai trong v ng 60 n 120 ngy. Trong khi khng p d ng cc bin php gy ng dc ng lot, t l chm sinh l rt cao: 56,8% s b c cha li sau trn 150 ngy.

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325

Bng 1: T l b ng dc, phn b v kt qu u thai sau th tinh ln th nht

Ch s Quy trnh S lng b s dng S ng dc ln u S ng dc ln sau S b khng ng dc Phn b S b ng dc sau 24 h S b ng dc sau 48 h S b ng dc sau 72 h S b ng dc sau 96 h S b ng dc sau 120 h C cha sau dn tinh I I 28 13 17 11 II 24 6 (25) 18 (75) 6 (25) nh lng (n ; %) III 31 8 18 13 IV 32 0 32 (100) 0 V 30 11 (36,7) 21 (70) 9 (30)

(46) (61) (39)

(26) (59) (41)

1 (3,5) 5 (17,9) 5 (17,9) 3 (10,7) 3 (10,7) 12 (70,6)

0 4 (16,7) 8 (33,3) 3 (12,5) 3 (12,5) 9 (50)

0 6 (19,3) 10 (32,3) 2(6,4) 0 13 (72,2)

0 19 (59,4) 13 (40,6) 0 0 32 (100)

0 4 (13,3) 15 (50) 2 (6,7) 0 21 (100)

Bng 2: Khong thi gian t lc b n lc phi c cha li

Cc thng s S b (con) Khong t lc b n phi c cha (ngy) Min., Max., (ngy) 69 91 + 28 35 - 180 ngy n th nghim (quy trnh V) 389 214 + 152 35 - 608 ngy Tng n

Phn b: < 60 ngy 61 - 90 ngy 91 - 120 ngy 121-150 ngy > 150 ngy

10 22 27 9 1

(14,5%) (31,9%) (39,1%) (13,1%) (1,4%)

21 56 50 41 221

(5,4%) (14,4%) (12,9%) (10,5%) (56,8%)

KT LUN Tim kt hp PGF2 v GnRH cch nhau 7 ngy, theo di ng dc dn tinh l quy trnh ti u, cho t ng dc v rng trng ng pha v t l u thai cao. p dng quy trnh gy ng dc v rng trng ng pha c th l m 85% s b sa c cha li trong vng t 1 n 3 thng sau khi b . TI LIU THAM KHO 1. 2. De Rensis F., Peters A. R., 1999: Reprod. Dom. Anim., 34: 49-59 Gong J. C. et al., 1996: Biol Reprod., 55: 68-74

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3. 4. 5. 6. 7.

Kastelic J. P., 1994: Vet Med., 47: 64-71 Lauderdale J. W. et al., 1974: J. Anim. Sci., 38: 964-967 Pierson R. A. and Ginther O. J., 1988: Anim. Reprod. Sci., 16: 81-95 Pursley J. R. et al. 1995: Theriogenology, 44: 915-923 Savio J. D. et al., 1988: J. Reprod. Fert, 88:569-579

SUMMARY

Modification and adaptation of the estrus -ovulation synchronyzation techniques in the hostein -friz cows for shortening anoestrus period and calving internal
Le van Ty(1), Hoang Nghia Son(2), Nguyen Mong Hung(3) (1)Institute of Biotechnology (2)Institute of Tropical Biology (3)Univ Natl Science, Hanoi

Five modifications of the treatment with PGF2 and GnRH for oestrus synchronization in dairy cows have been tested an d compared. High percentage of cows became estrus synchronized with ovulation and high concepted rate after IA were recorded in the cows received 2 injection of PGF2 (2 ml) mixed with GnRH (10?g) with interval 7 days apart. Application of this treatment for 65 post-partum cows immediately a month after calving, reduce d the interval from paturation to the conception to 91 + 28 days against 214 + 152 days in the cas of routine estrus detection and IA. So treatment with 2 injections of PGF2 mixed with GnRH not only made oestrus synchronization more feasible but could be used for shortening the length between two parturation in cows.

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KCH THC CA QUN TH NANG NGUY N THU V PHN NG TR LI S KCH THCH GY RNG NHIU TRNG TRU V B
Hong Ngha Sn, Vin Sinh hc Nhit i L Vn Ty, Vin Cng ngh Sinh hc Nguyn Mng Hng, HKHTN, H Ni

M U u th ca vic to phi bng ph ng php gy rng nhiu trng th hin c tnh l cc phi ny c s c sng gn ging nh phi do rng trng t nhin: trong iu kin cy phi ti ng pha, t l cy phi t gn vi t l th tinh nhn to (xp x 70%); phi thu c do gy rng nhiu trng cng c kh nng sng t ng t nh phi thu c do rng trng t nhin sau khi ng lnh v gii ng. Nu i vi b, vic to phi bng gy rng nhiu trng c th p ng c cc chng trnh ci to ging bng cy phi th tru, cu tr li vn cn ng. Vic pht hin cc sng nang trong chu k sinh dc ca tru v ca b cho php ngi ta rt ngn khong cch ti s dng ng vt cho vic gy rng nhiu trng v thu phi. iu ny c chng minh rt hu ch tr n b. Cng c quy lut pht trin nang kiu sng nh b nhng hiu qu ca vic gy rng nhiu trng tru vn ch a c khng nh. iu g to cho tru cc khc bit nh vy? cc cng trnh trc, khi so snh qun th nang, chng ti thy rng tru c s nang c xoang ch bng 20% so vi b [1]. Ngoi ra, b, ngi ta pht hin thy v mt s l ng qun th nang c xoang t l thun vi qun th nang nguyn thu [2]. Tuy nhin, theo la tui, qun th nang nguyn thu ca bung trng c bin ng rt ln, hiu ng tui c th l m lu m hiu ng c th hoc hiu ng lo i. Vic nghin cu qun th nang nguyn thu ca bung trng tru v b giai on pht trin bo thai (t 2 thng n 9 thng tui), giai on sm nht ca qu trnh hnh thnh v pht trin, mt mt xo i khong trng tr ong hiu bit ca chng ta v qun th nang ca tru giai on n y, mt khc, so snh v mt nh lng, s l hu ch cho vic tm ra nguyn nhn phn ng thp tru trong vic tr li cc hooc - mn gy rng trng. VT LIU V PHNG PHP Bung trng ca bo thai tru v bo thai b c thu mt cch ngu nhi n ti hai tnh Ph Th v Bc Kn

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Bung trng c x l theo phng php nghin cu t chc hc thng thng [4], c m t ngn gn nh sau: sau thi gian c nh, t nht l 1 thng, ton b bung trng c lm kh bng cc nng cn tng dn t 400, 700, 900 ri n cn tuyt i, thi gian i vi mi nng t nht l trong 1 gi. T y bung trng c chuyn qua dung mi to-lu-en 2 ln, pa-ra-phin 50%, toluene + pa-ra-phin 80% ri chuyn vo c khi. dng ny, bung trng c th bo qun c lu cho n khi tin hnh ct mu. Ct ton b bung trng bng my ct mu sao cho cc lt ct d y 7? to thnh bng lin tc. Cc bng ny c 370C trong 2-4 ngy. C 6 lt ct lin tc li ly mt lt a ln lam knh, loi b pa-ra-phin, nhum bng haemtoxylin v phn tch di knh hin vi. xc nh s nang nguyn thu ca cc bung trng bo ca thai tru v b, tin hnh m s nang khi nhn thy nhn ca t bo trng, c 60 lt ct, li tin hnh m 1 ln, s nang nguyn thu trong mi bung trng c tnh theo cng thc: P = A x M/L + M Trong : P S nang bung trng; A - Tng s nang m c; M - dy ca lt ct; L - ng knh ca nhn t bo trng i vi mi bung trng, cn m lp li 2 ln; kt qu cui c ng l trung bnh cng ca 2 ln m ny.
Bng 1: S mu bung trng ca b o thai tru c phn tch t chc hc
Chiu di ca thn bo thai (cm)
8-14 15-20 23-26 34-47 50-60 63-70 80-85 89 Cng:

Tui ca bo thai (thng)

<3 4 5 6 7 8 9 10

S mu (n)
4 1 5 6 5 3 3 1 28

Bng 2: S mu bung trng ca b o thai b c phn tch t chc hc

Chiu di thn bo thai (cm)
12 18-19 24-26 34-38 45-47 55-57 65 Cng:

Tui ca bo thai (thng)

2,5 3 4-5 6 7 8 9

S mu (n)
1 3 4 3 5 4 1 21

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KT QU V THO LUN bung trng ca b o thai tru, cc du hiu phn bo gim phn ch bt u quan st c khi bo thai bt u chuyn sang thng pht trin th 3, th 4; tuy vy phn bo nguyn phn ch hon ton ngng vo cui thng th 6. Qun th nang nguyn thu hnh thnh chnh vo giai on ny. Vo thng th 4, quan st thy xut hin cc nang c xoang, tuy nhi n c nhiu nang ch cha rt t t b o nang v nhanh chng thoi ho. Nang c xoang xut hin nhiu v c cu trc c trng t thng th 6 tr i. Cng t thng th 6 tr i, qun th nang nguy n thu c xu th gim dn theo tui ca b o thai tru (bng 3), cng nh b (bng 4). Nu t l thoi ho ca nang b o thai tru v bo thai b l xp x bng nhau v cng c xu hng gim i theo tui ca b o thai th kch thc ca qun th nang nguyn thu ca bung trng tru lun nh h n so vi bung trng b mi thi im. So snh bt i xng gia qun th nang ca tru v qun th nang ca b thy r rng iu ny: thng th 7 l 159.540 - 94.930 so vi 341.260 - 110.050, (P=0.05) v thng th 8 l: 94.500 - 30.150 so vi 169.550 - 64.130 (P<0.05) Theo di qu trnh pht tri n c th ca tru v s bin ng ca kch thc qun th nang cho thy qun th n y kh n nh trong giai on chuyn tip t i sng phi sang giai on ngh, sau th gim rt nhanh t giai on ngh n giai on tr ng thnh. Quy lut ny cng quan st thy b. Vic nghin cu nh lng qun th nang nguyn thy ca tru v b giai on i sng phi cho php khng nh l : kch thc ca qun th nang nguy n thu ca bung trng tru l nh hn so vi kch thc qun th nang nguyn thu ca bung trng b ngay trong giai on i sng bo thai. Mc d c s dao ng c th rt ln, s nang xc nh c trong bung trng b l ph hp vi nhng cng b trc y [5]. Trong khi , s nang trung b nh trong bung trng ca tru cc thng cui ca giai on i sng b o thai, giai on ngh v c bit giai on trng thnh, thp hn rt nhiu so vi ca b cc giai on tng ng. iu ny cho php kt lun l ngay t khi hnh thnh, qun th nang ca bung trng tru t hn so vi ca b. tru v b trng thnh, u tm thy tng quan ng bin gia qun th nang nguyn thu vi qun th nang c xoang [2]. iu n y c ngha l mt qun th nang nguyn thu ln tng ng vi mt qun th nang c xoang ln. iu n y khng ch ng vi mi c th trong phm vi mt lo i m cng ng nu so snh gia hai loi tru v b. S nang c xoang ca b ln gp 5 ln s nang c xoang ca tru, tng ng vi qun th nang nguy n thu ca b cng ln hn nhiu so vi ca tru. L thuyt n y c kim nghim khi nghi n cu nh lng trc tip qun th nang tru b giai on bo thai.

330

Hi ngh KHOA HC V CNG NGH 2007

Bng 3: S hnh thnh qun th nang trong bung trng tru

L (cm) 8-14 15-20 23-26 34-47 50-60 63-70 80-85 89 T (thng) 3 4 5 6 7 8 9 10 n 4 1 5 6 5 3 3 1 MI +++ ++ +/ME +/++ ++ +++ 863.380* 151.250-86.340 159.540-94.930 94.500-30.150 61.130-34.020 56.350 35,2-11,4 27,6 - 9,8 11,8 - 5,2 14,0 -8,4 8,2 - 2,5 +/+ ++ ++ +++ D tr nang Thoi ha nang (%) NCX

Ghi ch: * Ch m c 1 mu; L. chiu di ca bo thai; T. tui ca bo thai; MI. nguyn phn, ME. gim phn, NCX. nang c xoang; n. s mu.
Bng 4: S hnh thnh qun th nang trong bung trng b
L (cm) 12 18-19 24-26 34 45-47 55-57 65 T (thng) 2.5 3 4-5 6 7 8 9 n 1 3 4 3 5 4 1 MI +++ ++ +/ME +/++ ++ +++ D tr nang ** ** 1.743.000+353.000* 648.000+260.000 341.260-110.050 169.550-64.130 153.270 38,6 - 11,5 15,0 - 8,5 +/+ ++ +++ Thoi ha nang (%) NCX

Ghi ch: ** khng m c; * ch m c 2 mu; L. chiu di ca bo thai, T. tui ca bo thai; MI. nguyn phn; ME. gi m phn; NCX. nang c xoang; n. s mu.

Cn c vo thc t gy siu bi non hai loi ng vt ny cho thy: tru c s nang c xoang ch bng 1/5 s nang c xoang ca b . Vi ngun huy ng nang t d tr nang nguyn thu thp hn nhiu so vi b, tru cho s trng rng v s phi tng ng trong mi ln x l si u bi non nh hin trng l hon ton hp l. Suy lun ny cng cho php t du chm cho cc c gng ci tin k thut si u bi non nhm ci to di truyn n tru. iu l th l cho d kch thc ca qun th nang nguyn thu gim i theo tui, c tru v b s lng nang c xoang l kh n nh [6]; iu ny cho php suy ra l c mt s nang nguyn thu n nh c huy ng vo qu trnh khi ln nang, hay ni cch khc s nang tham gia vo cc sng nang lun n nh. S khc bit v s nang c xoang gia tru v b c bn cht di truyn, do vy vi cc cng thc gy rng nhiu trng bng hooc - mn nh hin nay th vic trin khai cy phi ci t o con tru l khng kh thi. iu ny cng gi tm quan trng ca vic to phi in vitro i vi con tru.

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KT LUN Qun th nang nguyn thu ca bung trng tru nh h n qun th nang nguyn thu ca bung trng b ngay giai on hnh thnh. TI LIU THAM KHO 1. 2. 3. 4. 5. 6. Le Van Ty et al., 1989: Anim. Reprod. Sci. 19: 171 -178. L Vn Ty v cs, 1995: K yu Vin Cng ngh Sinh hc: 186-194, H Ni. Abbel-Raouf and El Naggar, 1974 : J. Reprod. Fert, 41: 478-483. Monniaux D. et al., 1983: Theriogenology, 19: 51-81. Erickson B. H., 1966: J. Anim. Sci., 25: 800 -825. Le Van Ty et al., 1994: Anim. Reprod. Sci., 35: 191 -199.

SUMMARY

The size of the primordial follicular population and the super ovulation response in buffaloes and cows
Hoang Nghia Son(1), Le Van Ty(2), Nguyen Mong Hung(3) (1)Institute of Tropical Biology (2)Institute of Biotechnology (3)Univ Natl Science, Hanoi

28 ovaries of swamp buffalo foetus and 21 ovaries of local yellow cow foetus in Vietnam have been collected, fixed in Boin -Holland and analysed by routine histologic technique. The pool of primordial follicles of the ovaries have been determined and compared. The size of the pool of primordial follicles of the ovaries of buffaloes was smaller than that of the cows in every recorded foetal development periods. That indicated the faible response of the superovulation in buffalo. It could be a consequence of poor population of the antral follicles due to small pool of primordial follicles.

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THU NHN V BIT HO T BO GC TRUNG M T TU XNG CHUT (Mus musculus var. Albino)
Trng nh, Trng Hi Nhung, Phm Vn Phc, Phan Kim Ngc H Khoa hc T nhin TPHCM

M U Tu xng cha mt ngun t bo gc trung m (Mesenchymal stem cell -MSC) di do. Cc t bo MSC ny c thu nhn v nui cy trong mi trng DMEM/F12 (Sigma), 10% FBS (Invitrogen). Sau kho ng 10 ngy nui cy, cc t bo MSC tng sinh mnh v chim 70-80% din tch b mt y bnh Roux (25 cm 2, Nunc), tin hnh cy chuyn vi trypsin/EDTA (Sigma) 0,25% nhm cung cp cht din h dng v khng gian pht trin cho t bo MSC. T bo MSC l t bo gc a nng, chng c kh nng bit ho th nh rt nhiu kiu t bo chc nng khc nhau. Chng c th bit ho th nh t bo xng khi nui trong mi trng DMEM/F12, 10% FBS c b sung dexamethasone, glycerol phosphate, ascorbate, EGF (nhn t tng trng biu m); thnh t bo m khi nui trong mi trng DMEM/F12, 10% FBS c b sung isobutyl -methylxanthine, dexamethasone, insulin, indomethacin; thnh t bo ging t bo thn kinh khi nui trong mi trng DMEM/F12, 10% FBS c b sung dch chit no chut. T bo gc ni chung v t bo gc trung m ni ring hin ang l mi quan tm hng u ca nhiu nh khoa hc v nhng thy thuc lm sng bi kh nng c bit duy nht ca chng l kh nng bit ho thnh nhiu kiu t bo khc nhau trong c th, nh xng, sn, m, t bo thn kinh... Thm vo chng l mt ngun t bo y ha hn cho vic cy ghp v sinh dc phm, chng cng phc v nh l mt m hnh ti u ca s pht trin ca ng vt c xng sng. Trong cc nghin cu v s bit ho, cc t bo MSC bit ho thnh xng, sn v m c s dng nhiu nht nh l bng chng c th v tim nng bit ho ca t b o gc. VT LIU V PHNG PHP
Thu nhn tu xng chut

Chut (Mus musculus var. Albino) b git bng cch ko gi n t sng c, sau thu nhn hai xng i v xng cng chn. Cc khc xng c ra bng dung dch PBSA hai ln. Dng ko v kp tch b m c bm trn cc on xng. Cui cng, tu xng c thu nhn bng cch: dng ng kim vi mi kim 26 G c cha sn mi trng nui cy, chc xuyn vo tu xng v di ra hai ln.

Phn IV: CNG NGH SINH HC NG VT

333

Nui cy chn lc t bo gc trung m Hn hp t bo c trong tu xng bao gm t bo gc to mu, t bo mu trng thnh v t bo gc trung m. Tuy nhin, ch c t bo gc trung m mi c kh nng bm dnh vo b mt nha ca bnh Roux. Sau vi ln thay mi trng, ta c th loi b c tt c cc t bo tp nhim, ch cn li duy nht t bo gc trung m. Tin hnh nh sau: huyn ph t bo tu xng sau khi thu nhn c nui trong bnh Roux vi mt 1.104 t bo/cm2 iu kin 37 0C, 5% CO 2. Sau 24 gi, cc t bo gc trung m bt u bm trn b mt bnh Roux, thay mi trng loi b cc t bo khng bm, tip tc nui n khi t bo t mt 70-80% bnh Roux vi ch thay mi trng l 3 ngy/ln.
Cy chuyn tng sinh

Khi mt t bo MSC trong bnh nui t khong 70-80%, tin hnh cy chuyn tng sinh nhm cung cp khng gian v cht dinh dng cho t bo MSC tng sinh v pht tri n t hiu qu cao. Tin h nh nh sau: b mi trng c v ra t bo vi 4-5ml PBS-gentamycin (10 IU/ml) hai l n. Sau , b dch ra v b sung 4-5ml trypsin/EDTA 0,25%. Sau 15 giy, ti n hnh b dung dch enzyme nhng vn cha li khong 1ml v tip tc trong t m 37 0C trong 2-3 pht. Sau , lc nh bnh Roux tch t bo ra khi b mt y. Sau khi t b o trn ln v tch ra khi b mt nui, trung ho trypsin tha bng 10-11ml mi trng DMEM 10% FBS. Huyn ph t bo c chia u cho 3 bnh Roux mi.
Bit ho t bo gc trung m

Bit ha t bo gc trung m thnh t bo to m bng mi trng nui DMEM/F12 10% FBS b sung hn hp 1 M dexamethasone, 200 M indomethacin, 1,7 M insuline, 500 M isobutyl -methylxanthine (tt c u ca Sigma). S bit ha c ghi nhn khi quan st d i knh hin vi phng i X20, X40 thy c s xut hin cc git m nh. Ngoi ra, cc t bo m cn c xc nh da vo phng php nhum vi thuc nhum Sudan black (Merck) l thuc nhum lipid, n ch h a tan trong lipid v to mu en. Bit ho thnh t bo xng: Cc t bo MSC c nui trong mi trng DMEM/F12 10% FBS vi 100 nM dexamethasone, 50 g/ml L -ascorbic acid 2phosphat (AsAP) v 100 mM -glycerolphosphate (tt c u ca Sigma). Sau 7-14 ngy, nh gi s bit ho thng qua kh nng tch t ca calcium trong cht nn vi phng php nhum vi thuc nhum Alizarin Red (Sigma). Bit ho thnh t bo ging t bo thn kinh: Mi trng bit ho t bo gc trung m thnh t bo thn kinh l mi trng DMEM/F12 10% FBS, b sung 10% dch chit no chut. Xc nh t bo thn kinh sau khi c bit ho bng hnh dng c trng ca chng.

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KT QU V THO LUN
1. Nui cy chn lc t bo MSC

Sau 24 gi nui cy, t bo MSC c hin tng bm trn b mt bnh Roux, trong khi cc t bo to mu vn tri l lng trong dch huyn ph t bo.

(a)

(b))

(c)

(d)

Hnh 1. Kt qu nui cy chn lc MSC . (a) T bo MSC va mi thu nhn; (b) T bo MSC sau 24 gi nui cy, cc t bo MSC bt u bm dnh vo b mt bnh nui; (c) T bo MSC sau 72 gi nui cy, cc t bo MSC bt u tri di hnh dng v tng sinh; (d) Hnh nh cc t bo MSC sau 240 gi nui cy, hu ht cc t bo MSC lc ny c dng hnh thoi c trng v tng sinh mnh. ( phng i X20)

Sau 48 gi, loi b mi trng c v thay mi trng mi nhm cung cp cht dinh dng cho s pht trin ca t b o MSC. Khi thay mi tr ng, ng thi loi b c nhng t bo cht cng nh cc t bo to mu cn l lng trong mi trng c. Vi ln thay mi trng u tin, khng tin hnh ra t bo v kh nng bm dnh ca t b o MSC vo b mt bnh Roux vn cn yu. Sau 72 gi, t bo MSC bt u tri ra trn b mt bnh Roux, khi t bo MSC c hnh dng c trng, thng l hnh thoi. Sau 120 gi nui cy, t bo MSC tip tc tri rng ra v phn chia gia tng s lng trn b mt bnh Roux. Sau 240 gi, t bo MSC hp dng, bm u v tri rng trn b mt bnh Roux. Khi mt t bo MSC t 70% - 80% din tch bnh Roux, tin hnh cy chuyn nhm cung cp khng gian sng v cht dinh dng cho t bo MSC.
2. Cy chuyn tng sinh t b o MSC

Khi va c cy chuyn, t bo MSC cha c hnh dng c trng v tri l lng trong mi trng ging nh khi va c thu nhn t tu xng.

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Sau 24 gi, t bo MSC bt u bm dnh v tri rng. Sau 48 gi, t b o MSC tri rng, c dng hnh thoi c trng v bt u tng sinh. Tuy nhi n, kh nng tng sinh ca t bo MSC lc ny vn cn rt chm. Sau 152 gi, t bo MSC hp dng v tri u trn b mt bnh Roux.
Bit ho t bo MSC

X20

(a)

X40

(b)

X20

(c)

X20

(d)

Hnh 2. Kt qu bit ho t bo MSC. (a) Cc t bo to m: sau 7-14 ngy trong mi trng bit ho to m, cc t bo bt u tch t cc git m ( ); (b) Cc git m bt m u en khi nhum vi thuc nhum Sudan Black; (c) Cc t b o to xng: sau 7-14 ngy trong mi trng bit ho to xng, cc t bo bt u trn li v tch t calci trong cht nn. S tch t calci c xc nh bng cch nhum vi Alizarin Red; (d) T b o ging t bo thn kinh: cc t bo MSC sau khi nui trong mi trng bit ho thn kinh, MSC thay i h nh dng thnh t bo ging t bo thn kinh vi exon di ( ), thn t bo thn kinh ( ).

Bit ho hnh t bo m

Sau 48 gi, cc t bo bt u tch t cc git m trong t b o cht. Cc git m nh gp li dn thnh cc git ln. Chng chuyn t dng d i, tri rng chuyn sang dng trn. Cc git m c th quan st d d ng di knh hin vi o ngc c phng i X20. Bit ho hnh t bo xng Sau khi nui trong mi tr ng bit ha, cc t bo t dng di chuyn sang dng trn v hnh ht u. V mt hnh thi hc, c th nh gi s b l cc t bo ny bit ha thnh cng. Dng thun di, tri rng l hnh dng ca t bo gc trung m v dng trn hay hnh ht u l hnh dng ca t bo to xng (osteoblast). Khi nhum vi Alizarin red, cc t b o trn hay hnh ht u s nhum mu cam. iu ny chng t cc t bo c ngun gc t t bo MSC ny c s lng t mui calci trong cht nn ngoi b o. Nh vy, cc t bo MSC bit ha thnh cc t bo to xng.

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Bit ho thnh t bo ging t bo thn kinh Sau 10-14 ngy bit ho, mt s t bo bt u thay i hnh dng vi si trc ko di v mt u phnh to ging thn t bo thn kinh cng nh cc nhnh nh xut pht t thn ging nh cc si nhnh. KT LUN Thu nhn v nui cy thnh cng t bo gc trung m t ty xng chut Mus Muscular var. Albino. T bo gc trung m t ty xng chut Mus Muscular var. Albino c kh nng bit ha thnh t bo to m, xng v t bo thn kinh khi nui cy trong mi tr ng c b sung mt s nhn t cm ng bit ho thch hp. TI LIU THAM KHO 1. 2. 3. 4. Daniel R. Marshak, Richard L. Gardner & David Gottlieb (2001). Stem cell Biology. Cold Spring Harbor Laboratory Press. Freshney R. I. (2000). Culture of animal cells - A Manual of basic Technique, 4th Ed. Newyork: Willey Liss. Moustapha Kassen v Cs (2004). Mesenchymal Stem Cells: Cell Biology and Potenntial Use in Therapy. Basic & Clinical Pharmacology & Texicology, 95, 209-214. Robert I.(2004). Mesenchymal stem cell. Vox Sanguinis. 38-41.

SUMMARY

Collecting and differentiating mesenchymal stem cells from mouse bone marrow
Truong Dinh, Truong Hai Nhung, Pham Van Phuc, Phan Kim Ngoc University of Sciences HCMCity

Bone marrow is a rich source of mesenchymal stem cells (MSCs). MSCs have been collected and cultured with DMEM/F12 medium plus 10% FBS (fetal bovine serum). At about 10th day, MSCs strongly expand and cover with 70 -80% Rouxs surface. At that time, MSCs are subcultured by trypsin/EDTA 0,25% to provide nutrients and surface for development. MSCs are pluripotential stem cells. They can differentiate to many different cell types, such as: osteoblasts, adipocytes, neuron -like cells... MSCs could be differentiated to osteoblasts in DMEM/F12, 10% FBS medium plus dexamethasone, glycerol phosphate, ascorbate, EGF (epidermal growth factor); to adipocytes in DMEM/F12, 10% FBS plus isobutyl-methylxanthine, dexamethasone, insulin, indomethacin; to neuron -like cells in DMEM/F12, 10% FBS plus mouse brain crude extract.

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TO PHI B BNG PHNG PHP VI TIM TINH TRNG VO BO TNG TR NG

Trn Th Thanh Khng, ng Hong Lm, Phm Vn Phc, Phan Kim Ngc H Khoa hc T nhin TPHCM

M U nng cao hiu qu th tinh nhm nhn nhanh n b sa, vic p dng vi tim (microinjection) tinh trng vo bo tng tr ng l cn thit. Trng b thu nhn t nhiu ngun c nui trng thnh trong TCM 199 v i 20% FBS (Fetal Bovine Serum). Tinh trng b s a ng lnh c hot ho bng ph ng php swim-up trong mi trng BO. Sau khi vi ti m, trng c hot ho. Sau 9 ln thc hin vi 44 trng to, c 20 phi, vi t l th nh cng l 47,17% v phi t giai on morula l 10%. ICSI - Intracytoplasmic sperm injection l phng php vi tim m t tinh trng c la chn t trc vo bo tng trng, vi s h tr ca h thng vi thao tc cng vi knh hin vi o ngc. K thut ny ra i vo nm 1992 do Palemo v cs ng dng thnh cng trn ngi. ICSI nh l mt cuc cch mng trong h tr sinh sn c bit trong cha tr v sinh nam. i vi gia sc, sau khi thc hin ICSI, t b o trng c cha tinh trng phi tri qua qu trnh hot ho vi cc tc nhn nh xung in, ethanol 7%, Calcium Ionophore (A23187) To phi b bng phng php vi tim m ra hng mi trong nhn ging b em li hiu qu cao, c bit l b sa trong trng hp ngun giao t c cht lng km hoc ngun trng ng lnh. B n cnh , vic tip cn k thut hin i cn gip hon thin cc quy trnh cng ngh khc trong cc nghi n cu xa nh to dng, sinh thit, to ng vt chuyn gen, bo tn ng vt qu him VT LIU V PHNG PHP Chun b trng: Trng b ti thu nhn ti l m hay trng ng lnh c la chn, phn loi A, B. Cc trng n y c nui trng thnh trong mi tr ng TCM 199 (Sigma) v i 20% FBS (In vitrogen). Sau 24 gi nui, tin h nh ph lp t bo cumulus bao quanh tr ng bng enzyme hyaluronidase (1mg/ml, Sigma).

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(a)
Hnh 1. Trng b chn.

(b)

(a) Trc khi ph lp cumulus, (b) Sau khi ph lp cumulus

Chun b tinh trng: Tinh trng ng lnh trong cng r c gii ng 37 oC trong 60 giy. Thc hin swim-up 15 pht trong mi trng BO, thu nhn dch ni, chn tinh trng cho vi tim.

Hnh 2. Vi tim tinh trng vo bo t ng trng

Vi tim: Ht tinh trng bng kim tim (injection pipette) trong git PVP (In vitrogen) chuyn qua git TCM 199 cha trng. Kim gi (holding pipette) trng c iu chnh th cc v tr 6 gi hay 12 gi, tin h nh tim tinh trng vo bo tng trng mt cch nh nhng, nhanh chng vi th tch a vo bo tng trng t nht.

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Chuyn trng sau khi vi tim vo mi trng nui phi CR1aa. Sau 4 gi nui, tin hnh hot ho trng sau khi vi tim vi TCM 199 vi 7% ethanol trong 4 pht. Tip tc nui phi trong mi trng CR1aa v theo di cc giai on pht trin ca phi. KT QU V THO LUN Sau 9 ln vi tim, c 44 trng, s phi thu c l 20 phi, chim t l to phi l 47,17%. Trong 20 phi c 2 phi giai on 2 t bo (10%), 5 phi giai o n 4 t bo (25%), 10 phi giai o n 8 t bo (50%), 1 phi giai o n 16 t bo (5 %) v 2 phi giai on morula (10%).

(a)

(b)

(c)

(d)

Hnh 3. Kt qu to phi b bng ICSI.

(a) Phi 2 t bo, (b) Phi 4 t bo, (c) Phi 8 t bo, (d) Phi morula

Nm 1989, Younis v cs to c phi b bng k thut ICSI truyn thng v trng khng hot ha vi t l 2%. Li v cs (1999) to phi bng piezo-ICSI c hot ha bng ethanol 7% phi to ra vi t l 26,4%. Cng nm Hamano thnh cng vi t l tng i cao l 56,6 %. Nm 2004, Kato, H., Matsumoto v cs so snh gia hot ha bng ethanol 7% v khng hot ha, t t l phi l 51% so vi khng hot ha l 13%. Takenaka v cs (2005) cng d ng phng php hot ha bng ethanol 7% vi t l to phi l 75,6%.

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Nh vy vi t l to phi l 47,17 % l tng i cao, cao hn hn vi ln thc hin u tin vo nm 1989. So vi cc nghin cu gn y th t l to phi l tng i thp, tuy nhin cc nh khoa hc trn th gii thc hin ICSI c h tr Piezo trong khi chng ti thc hin vi phng php c in, dng lc c hc ca kim tim vo mng trng. K thut Piezo-ICSI, mng pellucida v mng non s chu t s tc ng ca u pipette m thay vo l c s h tr ca tia piezo. Mt trong nhng thun li ca k thut piezo l gim ng k s ph hy trng trong sut qu tr nh th tinh. Trong khi vi k thut c in, lc ca u kim gm lc tip tuyn v lc xoay phn no tc ng n t bo cht trng. Nh vy, vi k thut ICSI c in chng ti to ra phi vi t l 47,17% c ng vi s hot ha ca ethanol 7%. Tuy ethano l7% khng phi l tc nhn hot ha nhn to ti u nhng t l to phi cng tng i cao. Hn th na, ethanol 7% l ha cht d c v tin li trong iu kin chng ti C th khng nh rng vi ngun mu n nh, trng cht l ng tt, h thng vi thao tc tt, n nh cng vi tay ngh ca k thut vi n thao tc cao th t l phi b bng phng php ICSI khng ch dng t l 47,17% m cn cao hn na. i vi trng ngi, t l ICSI cao hn so vi IVF, nhng b: theo Kim tea Chung v cs (1999) t l phi IVF l 75%, ca Phan Kim Ngc v cs (2006) l 72,19% t l to phi bng ICSI thp h n do bn cht ca trng b kh hot ha hn so vi trng ngi v to thnh hp t, chng phi nhn nhng kch thch nhn to. Tuy nhin, Gyu-Jin Rho v cs (1998) tin hnh xc nh gii tnh tinh trng bng phng php Flow Cytometry vi t l 80% v tin hnh vi tim tinh trng xc nh gii tnh. T l phi t c vi phng php ny l 46,6% v phi giai on blastocyst l 6,9%. So vi kt qu to phi ca chng ti l 47,17 % cao hn. KT LUN Trong phng php chu n b giao t, swim-up dng cho hot ho tinh trng ph hp vi ICSI. Tc nhn ethanol 7% c vai tr hot ho trng sau khi vi ti m, cho kt qu to phi tng i cao v ph hp vi iu kin thc hin. Bng k thut ICSI c in, vi tc nhn hot ho l ethanol 7% t l to phi 47,17% vi phi t giai on morula l 10%. TI LIU THAM KHO 1. 2. 3. Carlos A. Guerrero. (2006) Cryopreservation And Intracytoplasmic Sperm Injection With Bovine Epididymal Spermatozoa . .S. Louisiana State University. Henry E. Malter & Jacques Cohen.(2002) Intracytoplasmic sperm injection: technical aspects. Gamete source, manipulation and disposition. Heuwieser W, Yang X, Jiang S, Foote RH: Fertilization of 21. Yanagida K, Katayose H, Yazawa H, et al(1992): Successful fertilizabovine oocytes after microsurgical injection of sp ermatozoa Theriogenology

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4.

Jin-Tae Chung (1999). Activation of bovine oocytes following intracytoplasmic sperm injection (ICSI). Department of Animal Science Macdonald Campus McGill University, Montreal, Quebec, Canada. Keith J. Betteridge (2004). New reproductive Technologies in Cattle: A Veterinary Perspective. Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, N1G 2W1, Canada. Kimura, Y & R. Yanagimachi (1995). Intracytoplasmic sperm injection in the mouse. Biol. Reproduction. 52: 709 -720. Mapletoft RJ & J. F. Hasler (2005). Assisted reproductive technologies in cattle: a review. (1) Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada (2) AB Technology, Bionich e Animal Health USA, Inc., Pullman, WA 99163, United States of America. Ragaa Mansour (1998). Intracytoplasmic sperm injection: a state of the art technique Human Reproduction Update , Vol. 4, No. 1 pp. 43-56. Toshitaka Horiuchi (2002) Application of Intracytoplasmic Sperm Injection to Animal Production. Faculty of Life and Environmental Sciences Department of Life Sciences.

5.

6. 7.

8. 9.

SUMMARY

Bovine embryo production by intracytoplasmic sperm injection

Tran Thi Thanh Khuong, Dang Hoang Lam, Pham Van Phuc, Phan Ki m Ngoc University of Sciences HCMCity

Due to bovine oocyte source is rare. To increase fertilization effect, application of intracytoplasmic 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 serum) until maturation. Cryopreserved bovine semen is thawed and activated with BO medium. After injection, oocytes are treated with 7% ethanol in TCM 199 for 4 min for activation. In 9 repeats, 44 oocytes were in jected, with 20 embryo that are formed, with 47,17% cleavage rate, 10% morula embryo.