PHA'M LE SO^ - Anh hu&ng ciia tdn sudt sieu dm ...
ANH H i r d N G CUA TAN SUAT SIEU AM DEN KET QUA THU TE BAO TRU'NG T f BO SONG DE TAO PHOI IN VITRO
(Ket qud bu&c ddu Ihuc hien nam 2006) . .. .
Phan LeSffn'', Nguyen Vdn Lf, Nguyen Thi Thoa\
Nguyen Khdnh Vdn^ Vu Ngpc Hi?u' vd Liru Thj Nggc Anh^
'B9 mon Sinh ly Sinh h6a v^ Tap tinh vat nuoi
^Ph6ng Thi nghifm trgng diem Te bio dpng vat
*Tac gia lien he: Phan Le Scm - Bo mon Sinh ly Sinh h6a va Tap tinh vat nuoi Vien Chan nuoi - Thuy Phucmg - Tir Liem - H& Noi
Tel: (04) 38.385.940/0914.551.149; Email: [email protected] ABSTRACT
Effect of ovum pick up frequence on oocyte number and embryo production
Phan Le Son*, Nguyen Van Ly, Nguyen Thi Thoa, Nguyen Khanh Van, Vu Ngoc Hieu and Luu Thi Ngoc Anh One experiment aiming at examining the possible effect of two frequences of ovum pick up (one time vs. two times/week) on oocyte number and embryo production was undertaken. Ovum pick up was conducted using non-surgical method with the ultra sound equipment. It was found that, there was no significant differences in number of oocytes obtained between two frequences (5.1 vs. 5.28). However, there was a significant differences in number of embryos at morular and/or blastocyst stages between two frequences of ovum pick up (38.83% vs.
42.48%).
Keywords: Ovum pick-up, oocyte number, embryo production, TTON
DAT VAN DE
San xuat ph6i in vitro bang ngu6n te bao tning khai thac tu bo song la mgt trong nhirng phuang phap mai nang cao hieu qua c6ng nghe ph6i. Phuang phap sieu am thu te bao tning cho phep thu duge nhiing te bao tning chua thanh thuc tir nhirng gia siic cai da dugc biet ro ngu6n g6c. Khi sieu am thu te bao tning lap lai ket hgp vai viec cai tien ky thuat tao ph6i in vitro co the khai thac nhieu lan de tan dung t6i da tiem nang sinh san cua con eai, dac biet la nhung con co gia tri di truyen cao.
Phuang phap sieu am hiit te bao tning tren bo s6ng va tao ph6i in vitro lan dau tien dugc nghien ciiu tai Vien Chan nu6i tir nam 2005. Day la phuang phap tien tien, doi hoi thiet bi sieu am dat tien va trinh do cao cua nguai thao tac. Ket qua thu te bao triing va chat lugng te bao tning thu dugc phu thugc vao nhieu yeu t6. De nang cao hieu qua ky thuat sieu am thu te bao triing va tao ph6i in vitro, chiing t6i da tien hanh de tai: Nghien cicu dnh hu&ng ciia khodng cdch sieu dm den ket qud thu te bdo trirng tic bd sdng de tgo phdi in vitro.
Nham xac dinh khoang each sieu am hiit te bao tning cho so lugng va chat lugng te bao tning la t6t nhat.
VAT LIEU VA PHirONG PHAP NGHIEN ClTU Vat lieu nghien cuu
G6m 5 bo lai huong sira (F2, n=2; F3, n = 3), co nang suat siia tren 5000 lit/chu ky, thl trgng
> 400kg. Nhirng bo nay dugc cho an 40kg co tuai va 4kg thiic an tinh/ngay va dugc nu6i nhit tai Tram nghien ciiu thir nghiem thiic an chan nu6i - Vien Chan nu6i.
Tinh cgng ra HF nhap tir My co boat lire tren 40%, co nang suat siia tren 10.000 lit/chu ky.
Loai tinh nay da dugc kiem tra nang suat qua dai sau. Bo nhan ph6i la bo Lai Sind va bo lai
VIEN CHAN NUOI - Tgp chi Khoa hgc Cdng ngh? Chan nudi - So 21-Th4ng 12-2009
huong siia (F2, F3) co thl trgng >250kg, co chu ky sinh ly sinh san binh thuang va kh6ng mac cac benh ve duong sinh due.
Noi dung nghien cuu
Xac dinh anh huong cua tin suat sieu am den s6 lugng te bao tning thu dugc Xac dinh anh buong cua tin suat sieu am den ket qua tao ph6i in vitro.
Phuong phap nghien cuu
Nbtmg con bo tit da dugc chgn lira ve mat di truyen se dugc sir dung may sieu am hoac kham qua true trang dl xac dinh kha nang cho te bao tning, tir do tien hanh thu te bao tning. Hut te bao tning dugc thuc hien m6i tuan mgt lan va 2 lan.
Qud trinh hut te bdo tricng duac tien hdnh nhu sau: , f ; :.;!•{ .••" J.. •-.! ;• J',-' i
Cho bo vao gia c l dinh, liy hit phan ra, rira sach, sat khuan am hg va khu vuc xung quanh.
Chuan bi he th6ng may sieu am, n6i dau do am dao vao may sieu am. N6i kim hiit te bao triing (kim 16G dai 55 cm), dat may tao ap suat a 120 mmHg, tuang duong t6c do dong chay 25 ml/phut. Chuan bi m6i truang hut te bao trimg (mDPBS ciia Sigma) co b6 sung 50 iu/ml heparin va 5% huylt thanh be va khang sinh (100.000 UI penicilin/ml + lOOmg streptomycin/ml) duy tri a nhiet do 37°C bang may on nhiet. " - " *
Nhe nhang cho dau do sieu am co gan he th6ng dan kim vao am dao, cho tay qua true trang xac dinh vi tri cua bu6ng tning, vi tri cac nang trimg r6i dua buong tning ve phia dau quet cua dau do, quan sat cac nang trimg tren man hinh may sieu am, dem s6 nang tning co mat tren buong tning, Truac khi hut dich nang tning, hiit mgt it m6i truong hiit trimg de lam tron he thong kim va 6ng dan. Sau khi chgc hiit 2 - 4 nang tning, nit kim ra, hiit dung dich thu te bao trimg de te bao tning chay vao 6ng falcon, tien hanh hut lap lai den khi kh6ng con nhin thay nang trimg nao tren bu6ng trimg thi chuven qua bu6ng tning d6i dien. Dich hiit dugc thu vao ong ly tam 50ml va duy tri a nhiet do 37 C bang may 6n nhiet. .,
Sau khi hiit xong mgt bu6ng trimg, dua ngay ve phong thi nghiem, lgc bang c6c lgc Emcon co duong kinh 16 lgc 20)j,m, 10ml dung dich con lai dugc chuyen vao dia petri v6 triing co duong kinh 90mm de soi tim te bao tning duai kinh hien vi soi n6i. Te bao trimg thu dugc dugc danh gia phan loai theo phuong phap cua Loos va cs (1989). Sau khi danh gia phan loai, te bao tning duge nuoi thanh thuc trong m6i truang TCM-199 co b l sung 5% huylt thanh be mai sinh trong 24 gia, tiep theo chiing dugc chuyin qua m6i truong thu tinh BO vai mat do tinh triing 6,25x10 /ml trong 5 gia.
Sau qua trinh thu tinh, hgp tir dugc tach te bao cumulus va chuyin sang nu6i trong m6i truong nu6i CRl c6 b6 sung axit amin, huyet thanh be mai sinh va albumin huylt thanh bo. Sau 7 ngay nuoi, nhiing ph6i dau, ph6i nang c6 chit lugng tit dugc lira ehgn dl ciy cho bo nhan dgng due d6ng pha hoac d6ng lanh bao quan. Sau 3 thang, kham thai qua true trang dl xac dinh ty le c6 chira.
X u l y so lieu
S6 lieu dugc xir ly theo phuang phap th6ng ke sinh hgc co sir dung phin mem Excel version 5.0 va Minitab Release 12.
Thoi gian va dja diem nghien cuu .' Thi nghiem dugc tien hanh tai Vien Chan nu6i tir thang 2/2006 din thang 12/2006
PHAN LE SO^ - Anh hu&ng cua tdn sudt sieu dm ...
KET QUA VA THAO LUAN ; Thu te bao trung tir bo song
Trong t6ng s6 276 lugt bu6ng tning bo s6ng dugc thu te bao tning bang phuong phap sieu am, sl tl bao tning thu dugc la 1112, trung binh thu dugc 4,03 te bao tning/buong tning/lan (Bangl).
Bang 1. Thu te bao tning tir bo s6ng
Chi tieu Ket qua
S6 lugt bu6ng tning sieu am .,,..• 276 . ;.
T6ng so te bao tning thu dugc 1112 Te bao tning thu dugc/bu6ng tning (Mean ± SE)/lan 4,03±1,41
S6 nang tning trung binh/bu6ng tning/lan trong thi nghiem cua chiing t6i thap hon so vai s6 lieu cua Rizos va cs. (2005) a ca bo hau hi va bo cai sau de (10,4 va 7,8 nang trimg/bu6ng tning tuong iing). Tuy nhien, s6 te bao tning binh quan thu dugc/bu6ng tning/lan trong thi nghiem cua chiing t6i kh6ng sai khac nhieu so vai ket qua cac tac gia nay (3,98 so vai 4,7 a bo hau bi va 2,8 a bo sau de).
Anh huong ciia khoang each thu te bao trung lap Iai
Kh6ng CO sir khac biet ve so lugng te bao tning thu dugc/bu6ng tning bo cai/tuan giira phuang phap sieu am mgt lan mgt tuan so vai hai lan mgt tuan. Vai khoang each sieu am mgt lin/tuan, s6 lugng te bao trimg trung binh thu dugc/bu6ng tning la 5,1. Trong khi do, a quy trinh sieu am 2 lan/tuan, s6 lugng te bao tning trung binh thu dugc la 5,28 (Bang 2).
Bang 2. Anh huong cua khoang each lap lai den ket qua thu te bao tning Khoang each
sieu am 1 lan/tuan 2 lan/tuan
S6 lugt bu6ng tning Dugc sieu am
156 120
Te bao tning thu dugc/tuan
n Mean 795 5,10 317 5,28 Ket qua sieu am thu te bao tning lap lai cua chiing toi con thap hon ket qua cua mgt s6 tac gia khac da c6ng b6. De Roover va cs. (2005) bao cao trung binh thu dugc 5,6 te bao tning/bu6ng tning/lan sieu am. Theo Reis va cs. (2002), tir 24 bo hau hi, bang phuong phap sieu am thu te bao tning lap lai, cac tac gia da tien hanh chgc hiit 8 lan trong t6ng s6 3234 nang tning va thu dugc 1241 tl bao tning (38,4%) tuong iing). Trung binh thu dugc 7,2 te bao tning/bo/lan sieu am thii nhat va 5,7 te bao tning/bo/lan sieu am lap lai.
Cac tac gia Viana va cs. (2004) da sir dung 2 quy trinh sieu am hiit te bao triing tir bo cai Gir va thay rang s6 lugng te bao tning thu dugc a quy trinh sieu am 1 lin/tuan la 8,9 te bao tning (chgc hiit tren 11,6 nang) lon ban a quy trinh sieu am 21an/tuan la 7,0 te bao tning (chgc hiit tren 12,1 nang) dieu nay tuong urng vai ty le thu te bao tning a quy trinh thii nhat la lan hon a quy trinh thii hai (74,3% so vai 59,0%). Nhu vay, a quy trinh sieu am 1 lan/tuan ty le thu te bao triing cua cac tac gia la cao hon cua chiing toi (74,3%) so vai 71,03%)) con a quy tririh sieu am thir 2lin/tuan ty le thu te bao tning cua cac tac gia lai cho ket qua thap hon cua chiing t6i (59,0%sovai71,03%o).
Phan loai te bao trung thu duffc tir bo song
Sau khi thu dugc, te bao tning dugc chgn lgc, phan loai dua vao chat lugng tl bao cumulus va
n.
400 , , . , , •-;
368 54 290 768 1112
%
:i-ii 35,97 33,09
4,86 26,06 69,06 100,0 VIEN C H A N N U O I - Tgp chi Khoa hgc Cdng ngh? Chdn nudi - S6 21-Thdng 12-2009
SU ding nhit cua khii nhan theo phuong phap ciia Loos va cs. (1989). Kit qua phan loai t l bao trimg dugc trinh bay a Bang 3.
Bang 3. Phan loai chat lugng te bao tning thu dugc Chat lugng Te bao tning thu dugc Loai A
Loai B Loai C Loai D
Loai sit dung (A,B) Chung
Bang 3 cho thay, co sir khac nhau ve ty le te bao tning thu dugc. Chat lugng te bao trimg loai A la cao nhit, chilm ty le 35,97%%, tilp theo la t l bao tning loai B va D (33,09% va 26,06%
tuong ung); ty le te bao tning loai C thu dugc la thap nhat, dat 4,86%). Nhu vay, te bao tning c6 the sir dung dugc dat 69,06%) (768/1112). Ty le te bao tning loai A va B ciia chiing t6i cao hon so vai ket qua ciia mgt s6 tac gia khac da bao cao.
Ty le te bao tning loai A va B cua chiing toi (60,39%©; 154/255) cao ban ty le te bao tning loai I va II ciia cac tac gia Viana va cs. (2004) khi sir dung quy trinh sieu am m6i tuan mgt lan (29,0%) va 2 lan mgt tuan (35%)). Tuy nhien, ket qua cua chung t6i con thap ban so vai ket qua cua Reis va cs. (2002) da bao cao. Theo cac tac gia nay, ty le te bao tning loai 1 va 11 thu dugc a lan sieu am 1 va 2 tuong iing la 54,9% va 46,8%o.
Anh huong ciia khoang each sieu am den chat luong te bao tning thu duac
Ty le te bao trimg loai A va B a tan suat 2 lan va 1 tuan/ lan kh6ng c6 sir khac nhau dang ke(35,3%; 35,65% so vai 35,0%; 32,07%, tuong iing). Nguge lai, te bao tning loai D a phuang phap thu te bao tning 2 lan mgt tuan thap ban so vai mgt lan mgt tuan (20,88% so vai 28,14%) (Bang 4).
Bang 4.
Khoang cac
1 lan/tuan 2 lan/tuan Chung
Anh
;h
huong cua I khoang each sieu am den chat lugn Chat lugng
Loai A Loai B n.
278 122 400
% n 35,0 255 35,3 113 35,16 368
% 32,07 35,65 33,85
g te bao trimg thu dugc te bao tning
Loai C n.
39 15 54
% 4,90 4,75 4,85
Loai D
n % 224 28,14
66 20,88 290 26,28 Ket qua thu te bao tning loai loai A, B ciia chiing a phuang phap thu te bao tning 1 lan mgt tuan (66,9%) va hai lan mgt tuan (74,3) cao hon ty le te bao tning loai I va II ciia cac tac gia Viana va cs. (2004) khi cac tac gia nay sir dung quy trinh sieu am hiit te bao tning mgt tuan m6tlin(29,0%o)va2linm6ttuin(35%). ' ' ;•'
Anh huong cua khoang each sieu am den ket qua tao phoi in vitro
Trong thl nghiem nay, chiing toi da sir dung 394 te bao tning dugc thu mgt tuan mgt lan va 153 te bao tning dugc thu hai lin mgt tuin dl nuoi thanh thuc, thu tinh va nuoi hgp tir phat trien den giai doan ph6i dau, ph6i nang. Ket qua cho thay kh6ng co sir khac biet ve ty le hgp tir phan chia giiia t l bao tning dugc thu mgt lin mgt tuin va t l bao tning dugc thu hai lan
PHAN LE SON - Anh hu&ng cua tdn sudt sieu dm ... ^
mot tuin (53,8% so vai 54,24%), nhung c6 sir khac nhau vl ty le hgp tir phat trien den giai doan phoi dau, ph6i nang (38,83%) so vai 42,48%)) (Bang 5).
Bang 5. Anh huong cua khoang each sieu am den ket qua tao ph6i in vitro Khoang
am 1 lan/tuan 2lin/tuan Chung
each sieu S6 te bao tning nu6i
394 153 547
Phat trien cua te bao tning Te bao trimg
N 212
83 295
phan chia
% 53,80 54,24 53,93
in vitro Te bao tning
n 153
65 218
phat trien
% 38,83 42,48 39,85 Ket qua nay ciing phii hgp voi nghien curu cua Viana va cs. (2004) da bao cao. Theo cac tac gia nay, ty le hgp tir phan chia giiia te bao tning dugc thu mgt lan mgt tuan va te bao trirng dugc thu hai lan mot tuan tuong irng la 68,8% so vai 68,6%), nhung co sir khac nhau ve ty le hgp tir phat trien den giai doan ph6i nang (31.8% so vai 21,6%, tuong iing)
Ket qua tao phoi va cay phoi tao ra in vitro
Tir 768 te bao tning loai A, B va C thu dugc tir bo s6ng bang ky thuat sieu am, da c6 547 (71,2%) tl bao tning thanh thuc, 366 (66,91%) t l bao tning thu tinh, 295 (53,93%) hgp tir phan chia va 218 (39,95%)) ph6i dau/ph6i nang loai A, B va C thu dugc. Trong 20 ph6i dau/ph6i nang loai A va B cay cho bo nhan dgng due d6ng pha, co 6 truong hgp eo chira, dat 30,0% va c6 5 be ra dai (Bang 6).
Bang 6. Ket qua tao ph6i va cay ph6i tao ra tir te bao tning thu tir bo s6ng Chi tieu Ket qua Te bao trimg dugc nu6i thanh thuc in vitro
Te bao tning thanh thuc in vitro
Te bao trimg thu tinh/te bao tning thanh thuc Hgp tir phan chia/te baa trimg thanh thuc Hgp tir phat trien/te baa tning thanh thuc Cay cho bo nhan dgng due d6ng pha (n) Bo c6 chira
Be sinh ra
Ket qua hgp tir phan chia trong nghien ciiu cua chiing t6i con thap hon so vai ket qua cua mgt s6 tac gia khac da bao cao khi sir dung te bao tning thu tir bo s6ng (Galli va cs. 2001: 64,4%);
Reis va cs. 2002: 81,1%; Rizos va cs. 2005: 80,8%). Tuy nhien, Ty le hgp tir phat triln trong nghien ciiu cua chiing t6i (39,85%) cao hon ty le cua cua Galli va cs. (2001) (25,39% ) da c6ng bo. Ty le co chira (30,0%) trong nghien cim cua chiing t6i con thap hon so ty le cua Galli va cs. (2001) da bao cao (55,4%).
n 768 547 366 295 218 20
6 5
%
71,2 66,91 53,93 39,85 30,33 25,0
KET L U A N V A D E N G H I
Ket luan
Kliong CO sir khac biet vl s6 lugng te bao tning trung binh thu dugc/bo/tuin giira phuang phap sieu am thu te bao tning lan mgt tuan so vai hai lin mgt tuin (5,10 so vai 5,28). Tuy nhien, co sir khac nhau v e t y le hgp tir phat triln din giai doan ph6i dau, ph6i nang giira phuang phap sieu am thu te bao tning lin mgt tuin so voi hai lin mgt tuin (38,83% so vai
VIEN C H A N N U O I - Tgp chi Khoa hgc Cdng ngh? Chdn nudi - S6 21-Thdng 12-2009
42,48%). Do vay de thu dugc nhieu s6 lugng te bao tning, tao ph6i, ciy ph6i va tang nhanh sl lugng dan be sinh ra co gia tri di truyen cao a khoang each sieu am hiit tl bao tning 1 tuan 2 lan cho ket qua t6t nhat.
De nghi
Tiep tuc nghien ciiu de tang s6 lugng te bao tning thu dugc/lan sieu am thu te bao tning.
TAI LIEU THAM KHAO
De Roover, R., Genicot, G., Leonard, S., Bols, P. and Dessy, F. (2005). Ovum pick up and in vitro embryo production in cows superstimulated with an individually adapted superstimulation protocol. Animal Reproduction Science 86: 13-25.
Galli, C; Crotti, G; Notari, C; Turini, P; Duchi, R; Lazzari, G. (2001). Embryo Production by ovum pick up from live donors. Theriogenology 55: 1341-1357.
Loos, C. Van Vliet, P.Maurik and M. A. Th. Kruip (1989). Morphology of immature bovine oocytes. Gemete Research 24: 197-204
Reis, A., Steines, M.E., Watt, R.G., Dohnan, D.F., McEvoy, T.G. (2002). Embryo production using defined oocyte maturation and zygote culture media following repeated ovum pick-up (OPU) from FSH- stimulated Sunmental heifers. Animal Reproduction Science 72: 137-151.
Rizos, D., Burke, L., Duffy, P., Wade, M., Mee, J.F., O'Farrell, K.J., MarSiurtain, M., Boland, M.P. and Lonergan, P. (2005). Comparisons between nulliparous heifers and cows as oocyte donors for embryo production in vitrro. Theriogenology 63: 939-949.
Viana, JHM. Camargo, LSA., Ferreira, A.M. (2004). Short intervals between ulfrasonographically guided follicle aspiration improve oocyte quality but do not prevent establishment of dominant follicle in the Gir breed of cattle. Animal Reproduction Science 84: 1-12.
*Ngu'6i phSn bifn! TS. Nguyen Thgc Hda; Ths.Trinh Vdn Thdn