VIEN C H A N N U O I - Tgp chi Khoa hgc Cdng nghe Chdn nudi - So 21-Thang 12-2009
ANH HUONG CUA TUOI BO DEN SO LUONG VA CHAT LU'ONG TE BAO TRlTNG CHOC HUT
B A N GSIEU AM
Phan LeSfftt', Nguyin Thi Thoa\ Luu Cong Khdnh', Chu Thi Yen', Nguyin Thj Huorng', Pham Khdnh Vdn^ vd Luu Thj Nggc Anh^
'BQ mon Sinh Iy Sinh h6a vd Tap tinh vat nuoi
^Ph6ng thi nghif m trong diem Te bdo dgng vat
*Tac gia lien h?: Phan Le Son - Bo mon Sinh ly Sinh h6a vd Tap tinh vat nuoi Vi^n Chdn nuoi - Thuy Phuang - Tir Liem - Hd N^i
Tel: (04) 38.385.940/0914551149 ; Email: sonphanle(§yahoo.com ABSTRACT
Effect of cow age on oocyte quantity and quality
Phan Le Son*, Nguyen Thi Thoa, Luu Cong Khanh, Chu Thi Yen, Nguyen Thi Huong, Pham Khanh Van and Luu Thi Ngoc Anh The aim of this study was to examin the possible effect of age of doner cows on oocytes quantity and quality.
Two groups of Holstein Friese cows of 3 and six years were used for this study. Ovum pick up was conducted twice a week. It was found that number of follicles with diameter of 2 to > 9mm obtained from 3 year old cows was higher than that from 6 year old cows (10.35 vs. 9.63) (P<0.05). Similarly, number of oocyte/ovary otained from 3 year old cows was higher than that from 6 year old cows (7.2 vs. 6.78) (P< 0.05). The number of oocytes qualified as A,B Grade, which can be use for maturation, fertilization and culture was higher in cows of 3 year old (5.39 vs. 5.12) than that in cows of 6 year old. It was conluded that the younger the cows were the greater the quantity and quality of oocytes were. . . . • . . • ,
Keyword: occyte, ovum pickup, follicle .•••'\'"::^.'• , "''•' •'-.•'•-'
DAT VANDE
So lugng, chat lugng, ngu6n g6c te bao tning dong mgt vai tro het sue quan trgng trong viec phat trien cac c6ng nghe sinh san nhu san xuat ph6i in-vitro, vi tiem, cloning, chuyen gen, thanh lap ngan hang te bao trimg,...Truac day te bao trirng de phuc vu nghien cim va san suat phoi bo a nuac ta dugc lay tir 16 mo nen chat lugng di truyen cua ph6i rat thap, kha nang trien khai vao san xuat rat ban che do a Viet Nam bo dugc giet thit a cac 16 mo chu yeu la bo vang, bo lai sind c6 kha nang di truyen va chat lugng te bao tning rat kem. Hien nay ky thuat sieu am hut te bao trimg tir bo song da thanh c6ng, cho phep chiing ta co dugc s6 lugng va chat lugng te bao trimg t6t ciia bo cho. Te bao trimg c6 gia tri di truyen cao, chu dgng dugc thai gian, do vay chat lugng phoi tot, de trien khai san suat, thu dugc san pham co chat luong cao, khai thac toi da tiem nang sinh san. Theo Hansel (2003) sieu am hut te bao trimg va thu dugc 10,4 te bao trimg/bo cho te bao trimg, vai 40% ph6i dau va phoi nang thu dugc sau khi nuoi chin va thu tinh ong nghiem thu dugc 4 phoi/tuan hay co it nhat 200 ph6i/b6/nam. Ket qua nay cao hon nhieu khi thu ph6i in vivo bang phuang phap gay rung tning nhieu 5,9 ph6i/lan hay 35 phoi/bo/nam (Thibier, 2001). Sieu am hut te bao tning chiu anh huong cua rat nhieu
\ cu t6 trong d6 tu6i bo cho te bao tning d6ng vai tro het siic quan trgng, lien quan mat thiet den sinh ly sinh san ciing nhu kha nang cho te bao tning. Nham xac dinh dugc miic do anh liuong cua tu6i bo den so lugng va chat lugng te bao tning thu dugc.
VAT LIEU VA PHUONG PHAP NGHIEN ClTU Vat lieu nghien ciru
4 bo sira HF thuin, co do tuli tir 3 (n=2) tuli va tir 6 tuli (n=2), co nang suat sua > 5000 lit sira/chu ky. Dugc nuoi duong va cham soc trong ciing mgt dieu kien chan nu6i, cho an 50 kg CO tuai va 4 kg thiic an tinh moi ngay.
PHAN LE SON - Anh hu&ng cua tudi bd ...
Noi dung nghien CUU ' ' ' ' Xac dinh anh huong cua tu6i bo len s6 lugng nang tning.
Xac dinh anh huong cua tu6i bo len s6 lugng te bao tning thu dugc. . , — - , . « Xac dinh anh huong ciia tu6i bo len chat lugng te bao tning thu dugc
Phuong phap nghien ciru
Chudn bi bo: Sau khi lap dat xong he th6ng may sieu am va nhiet do ciia m6i truong hiit te bao tning (trong binh 6n nhiet) dugc lam am len 37°C. Bo cho te bao tning dugc c6 dinh, gay te cue bg bang each tiem 4 - 6 ml lidocain 2% vao khau du6i, lay bet phan, rira sach va sat khuan khu am dao va khu vuc xung quanh bang c6n 70°.
Xdc djnh so luffng vd kich thudc nang trUiig
Cho dau gio sieu am vao true trang, co dinh buong tning, dua buong tning ve phia mat quet ciia dau gio sieu am. Nhe nhang xoay, dem va do kich thuae nang tning co mat tren buong tning qua man hinh ciia may sieu am. Qua trinh do, dem can thuc hien ti mi, chinh xac va ghi chep can than. . . -
'.I: • - - , - . . I . ; - • - : '
Hilt te bdo trung '^;
Sieu am hiit te bao tning dugc thuc hien sir dung kim 18 G dai 55 cm, ap suat 120 mmHg va tan suat 2 lan/tuan. M6i truong hut te bao tning (mDPBS cua Sigma) co bo sung 2%) heparin va 5% huyet thanh be va khang sinh (lOO.OOOiu penicilin/ml + lOOp.1 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 co dinh buong tning, dua cac nang trimg ve vi tri miii kim, quan sat cac nang tning tren man hinh may sieu am va chgc hiit dich nang tning. -
Trong qua trinh hiit he th6ng kim va 6ng dan lu6n lu6n chiia day m6i truong hiit te bao tning.
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 (giir am trong binh on nhiet 37''C), tien hanh hiit lap lai den khi kh6ng con nhin thay nang tning nao tren bu6ng tning thi chuyen qua bu6ng trimg d6i dien. ,-;
Lgc, soi tim te bdo trung u ' ;;i '. f >
Sau khi hiit xong mgt buong tning, dua ngay ve phong thi nghiem, lgc bang c6c lgc Emcon c6 duong kinh 16 lgc 20|im, khoang 10ml dung dich con lai dugc chuyen vao dia petri v6 triing CO duong kinh 90mm de soi tim, danh gia, phan loai te bao tning duai kinh hien vi soi n6i.
Ddnh gid chdt luffng te bdo trirng
Te bao tning thu dugc dugc danh gia phan loai theo phuang phap ciia Goodhand va cs.
(1999). Dua vao hinh dang, sir d6ng nhat cua te bao chat va dac biet la mang cumulus.
Loai A CO > 4 lap mang cumulus, phan b6 ro rang, lien ket chat che va te bao chat dong nhat.
Loai B CO > 1 lop mang te bao cumulus, lien ket chat che va te bao chat d6ng nhat.
Loai C mgt s6 phan vo ngoai te bao tning bi lot tran, te bao chat co lai kh6ng deu.
Loai D te bao tning hoan toan bi lot tran va te bao chat co lai kh6ng deu.
Xir ly so lieu
So lieu dugc xir ly theo phuong phap th6ng ke sinh hgc co sir dung phin mim Excel.
VIEN C H A N NUOI - Tgp chi Khoa hgc Cdng ngh? Chdn nudi - So 21-Thdng 12-2009
KET QUA VA THAO LUAN Anh huong cua tuoi bo Ien so Iuong nang trung
Nang trimg co mat tren buong truing
114 lugt bu6ng tning dugc sieu am a 2 nh6m bo thi nghiem vai 1137 nang tning co mat tren buing trimg. Trong d6 nang tning co kich thuae 2 - 8 mm va > 9mm ciia bo 3 tuli tuong iing 482 va 77 nang trimg, binh quan nang tning^u6ng trimg tuong iing 8,93 va 1,43 nang. 6 nh6m bo 6 tu6i nang trimg co kich thuae 2 - 8 mm va > 9m tuong iing 466 va 112 nang, nang tning^u6ng tning tuong iing 7,77 va 1,87 nang.
Bang 1. S6 lugng nang tning/bu6ng tning va s6 lugng nang tning CO kich thuae 2 - 8 mm, > 9 mm/bu6ng tning cua 2 nhom bo.
Tu6i bo cho te bao tning
3 tuoi 6 tu6i Chung
Nang trimg/bu6ng
tning Mean ± SE 10,35 ± 0 , 2 2 '
9,63 ± 0,29"
9,99 ± 0,36
Nang n.
482 466 948
tning 2 - 8mm Nangtrimg /buong trimg
Mean ± SE 8,93 ± 0 , 1 6 ' 7,77 ± 0,22"
8,31 ±0,15
Nang n
77 112 189
tning > 9 mm Nang tning /bu6ng trimg
Mean ± SE 1,43 ± 0 , 1 1 ' 1,87 ±0,17"
1,66 ±0,10 Cdc chic cdi a, b trong ciing cdt cd gid tri sd khdc (p<0,05)
Bang 1 cho thay, bo 3 tuoi co s6 lugng nang trung/bu6ng tning a kich thuae 2 - 8 mm cao hon nh6m bo 6 tu6i. Song a nang trirng co kich thuae > 9mm nhom bo 6 tu6i co phan cao hon. S6 nang trimg binh quan thu dugc cua chiing t6i kh6ng sai khac nhieu so vai so nang trimg trung binh/bu6ng tning cua Rizos va cs. (2005) a bo hau bi va bo cai sinh san tuong iing 10,4 va 7,8 nang tning/bu6ng trimg. Song lai cao hon Ramos va cs. (2009) khi tien hanh sieu am hiit te bao tning 2 lan/tuan va 1 lin/tuan sir dung hormon de kich thich thu dugc tuong img 7,05, 7,95 va 8,75 nang tning/bu6ng tning.
Nang trimg duffc hiit
Bang 2. T6ng s6 nang tning dugc chgc hut, nang tning dugc chgc hut/bu6ng tning
a 2 nhom bo.Tu6i bo cho te bao tning
T6ng s6 nang (2 - > 9 mm)
Nang tning hiit
dugc Nang tning dugc hiit/bu6ng tning
Ti le hiit
(Mean ± SE) 3
6 Chung
559 578 1137
454 472 926
8,41 ± 0,22' 8,00 ± 0,24*' 8,20 ±1,64
81,21 81,66 81,43 Cdc cha cdi a, b trong cimg cdt cd gid tri sai khdc (p <0,05)
Bang 2 cho thay, t6ng s6 nang tning dugc chgc hiit la 926 nang/ ting sl 1137 nang quan sat dugc chiem ti le 81,43 %. S6 lugng nang tning dugc chgc hut/bu6ng tning a ca 2 nhom bo la 8,20 nang. Kh6ng c6 sir khac nhau cua 2 nhom bo vl ti le nang tning dugc chgc hiit/nang tning quan sat dugc a nhom bo 3 tuli va 6 tuli, tuong iing 81,2 va 81,66 %. Sl lugng nang tning dugc chgc hiit/bu6ng tning cua chiing t6i cao ban so vai kit qua cua Manik va cs.
(2003) da nghien ciiu sieu am hiit tl bao tning a bo An co sl lugng nang tning dugc chgc
hut/bu6ng tning 4,25 nang tning. Goodhand va cs, (2000) chgc hiit dugc 6,65 nang
tning/buong tning.
PHAN LE SON - Anh hu&ng cUa tudi bd ... •
Anh huong cua tuoi bo Ien so luong te bao trung thu duoc
Sau 114 lugt buIng tning dugc sieu am hiit te bao tning co 796 te bao tning dugc thu, binh quan te bao tning/buing tning 6,98 te bao tning. Ti le thu dugc chiem 85,95 %/ t6ng s6 nang tning dugc chgc hiit. Co sir khac nhau ve te bao tning/ bu6ng tning thu dugc giira 2 nhom bo, a nh6m bo 3 tuoi thu dugc 7,20 te bao cao hon nhom bo 6 tu6i 6,78 te bao. Kh6ng c6 sir khac nhau ve ti le te bao tning thu dugc/nang tning dugc chgc hiit giiia hai nhom bo.
Bang Tu6i bo cho te bao trimg
3. Tong s6 te bao tning thu dugc, te Bu6ng tning
dugc hiit
Te bao tning thu dugc
bao trung/bu6ng Tebao tning/bu6ng
tning
tning
Te bao trimg thu dugc/nang tning dugc hut (%) (Mean ± SE)
3 6 Chung
54 60 114
389 407 796
7,20 ± 0 , 2 3 ' 6,78 ±0,13"
6,98 ±0,13
85,68
"• 86,22 85,95
Cdc cha cdi a, b trong ciing cdt cd gid tri sai khdc (p <0,05)
Nghien ciru de nang cao s6 lugng te bao tning dugc rat nhieu nha khoa hgc quan tam nghien cim de thu dugc t6i da te bao tning co chat lugng. Manik va cs, (2003) thu dugc 4,5 te bao/bu6ng tning. De Roover va cs, (2005) thu dugc binh quan 5,6 te bao tning/bu6ng tning khi tien hanh sieu am hiit te bao tning tir bo s6ng. Ket qua cua chung t6i ciing phii hgp vai Viana va cs, (2004) da thu dugc binh quan s6 te bao tning 7,0 te bao trung, Ramos va cs, (2009) thu dugc 7,05 te bao tning/bu6ng tning. ^. , .,,^ , , ,, Anh huong ciia tuoi bo len chat Iuong te bao trung thu duffc
Chat lugng te bao anh huong rat lon den kha nang phat trien sau khi nu6i chin, thu tinh, phan chia va phat trien den giai doan ph6i dau va ph6i nang. Sau khi chgc hiit xong, dich hut nang trimg te bao tning dugc dua ngay ve phong thi nghiem de lgc, soi tim va danh gia chat lugng theo 4 mirc do khac nhau (A, B, C va D). Chi co te bao tning loai A va B mai dii tieu chuan nuoi cay, thu tinh va tao ph6i. . , . , , , . ,
Bang 4. Te bao tning loai A, B, C va D cua 2 nh6m bo Tuoi bo cho te bao tning
3 6 Chung
Te bao tning loai A, B N
291 307 598
Te bao/bu6ng tning 5,39
5,12 5,25
Te bao trimg loai C, D n
98 100 198
Te bao/buong tning 1,81
1,67 1,74
Bang 4 cho thay, t6ng s6 te bao tning loai A, B la 598 te bao. Te bao trimg binh quan thu dugc/bu6ng tning cua nhom bo 3 tu6i 5,39 te bao, cao hon nhom bo 6 tu6i 5,12 te bao. Te bao tning loai C, D khong c6 sir khac nhau nhieu, tuong iing 1,81 va 1,67 te bao.
KET LUAN VA DE NGHI
Tuoi bo CO anh huong den s6 lugng va chat lugng te bao tning thu dugc.
Nhom bo cho te bao tning co do tu6i 3 tuoi cho s6 lugng va chat lugng te bao tning tot hon nhom bo 6 tu6i.
VIEN C H A N N U O I - Tgp chi Khoa hgc Cdng ngh? Chdn nudi - So 21-Th4ng 12-2009
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. 2005; 86: p. 13-25.
Dimitrios Rizos, Lisa Burke, Patrick Duffy, Mary Wade, John F. Mee, Kevin J.O'Farrell, Mairtin MacSiurtain, Maurice P. Boland and Patrick Lonergan, (2005). Comparisons between nuUiparous heifers and cows as oocyte doners for embryo production in vitro. Theriogenology 2005; 63: p.939-949.
Hasler J.F., Hasler W.B. Henderson, P.J. Hurtgen, Z.Q. Jin, A.D. McCauley, S.A Mower, B. Neely, L.S. Shuey, J.E. Stockes and S.A. Trimmer, (1995) Production, freezing and transfer of bovine IVF embryos and Halsel, B.M. Magunath, J.P. Ravinada, P.S.P. Gupta, M. Devaraj and S.Nandi(2003). Effect of breeding season
on in vivo oocyte recovery and embryo production in non - descriptive Indian river buffaloes. Animal Reproduction Science 2: 376-383.
K.L. Goodhand, M.E. Staines, J.S.M. Hutchinson and P.J. Broadbent, (2000). In vivo oocyte recovery and in vitro embryo production from bovine oocyte doner treated with progestagen, oestradiol. Animal reproduction 2000; 63; p.145-185
K.L. Goodhand, M.E. Staines, J.S.M. Hutchinson and P.J. Broadbent (1999). In vivo oocyte recovery and in vitro embryo production from doner aspirated at different frequencies or following FSH treatment.
Theriogenology 1999; 51, p. 951-961 ' ..
Manik, F.li, X.chen, W. pi, C. Lin and Z. Shi (2003). Collection oocytes through fransvaginal Ovum pick-up for invitro embryo production in Nanyang yellow cattle. Reproduction in Domestic Animal 42: 666-670 Ramos, R. Rupt, J.U. Camara, M. R. MoUo, I . Pivato, A. P. Marques and Satori. Effect of follicular wave
sunchronzation on invifro embryo production in heifers. Animal Reproduction Science 3: 201-207.
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 2005; 63: p.939-949.
Thibier.M, (2001). A constrated year for the world activity of the animal embryo fransfer industry. Newslett. 20, p.13-19.
Viana, JHM. Camargo, LSA and 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: p.1-12
*'^g\xb\ ^)\knh'\^n: TS. Phan Vdn Kiim; TS. Ddo Dire Thd - . ^ 0 ;