Nguyen Van Lam vd Dtg Tap chl KHOA HOC & CONG NGHE n2(12)/2: 149- 154
ANH Hl/dNG CUA KICH THlTdC NANG TRiTNG DEN CHAT LlTpNG TRlTNG LON NUOI THANH THVC IN VITRO
Nguyen Vdn Lam', Hiia Nguyft IMai^", Nguyin Vdn Hanh', Nguyin Vi^t Linh' ' Vi^n Cong nghi sinh hgc - Vi^n Khoa hgc vd Cong ug/ip Vi^l Nam
^Tru-dng Dgi hgc Khoa hgc - DH Tlidi Nguyen TOM TAT
Trong nghien ciru nay, chung toi khao s^t ty 1^ cac nhdm nang c6 kich thudc kh^c nhau c6 tren be mat buing tning Ign vd danh gid anh hudng cua kich thudc nang trung tdi chdt lugng trCrng trong qud trinh nuoi thdnh thyc in vitro. Cdc nang trdng dugc chia ihanh 3 nhom vdi kich thudc dudi 2mm, tir 2-3mm vd tren 3min vdi tylp Idn lupt la 60,8±3,1%; 26.l±2.0% va 13,1±2,5% (P< 0,05). Ty 1? trtrng logi A d nhom nang tren 3 mm cao han so vdi nh6m nang 2-3mm (20,5±6,8% va 5,8±2,3%), (P<0,05) trong khi nhom nang dudi 2mm khong c6 trimg logi A. Tning loai B d hat nhdm nang tir 2- 3mm va tren 3mm co ty le tuong duang nhau (63,1%±4,9 va 62,5±7,8%) con d nhom nang dudi 2mm thdp han chi dat 40,5±4,1% (P<0,05). NhOng trung dii tieu chuan dcac nhom nang dugc nuoi trong dilu kien 38.5 °C vd 5% COi. KII qud cho thay ty le trimg thdnh thuc d nhom nang tren 3mm cao nhat 60.7±5.4%, nhom nang tir 2-3mm vd dudi 2mm c6 ty le thdnh thuc thap hon (29,7±4,6% va 6,8±3,1%): (P<0.05). Kich thudc nang trimg c6 dnh hudng din ty le cua tirng loai trirng khi thu nhgn va ty le triing thdnh thuc sau qud trinh nuoi in vitro. Nang co kich thudc tren 3mm cho ket qua cao nhat ca vl so trimg tit vd ty 1^ trung thanh thyc sau qud trinh nuoi.
Tti' khoa: Kich thudc nang: Trirng Ign; Chdt lugng; Nudi thdnh thuc DAT VAN DE
Lgn la ngudn cung cap thyc pham chii yau cho con ngudi. Lgn cdn la ddi tugng gdn giii vdi con ngudi ve mat sinh ly hgc do dd lgn cd tiem nang trd thdnh ngudn nguyan iieu cac loai ndi tgng phuc vu cho y hgc thay the. Vi vay, Ign dugc quan tam nghien ciru dac biet tren phdi. Viec san xuat phdi Ign in vitro lam nguyen lieu cho cdc nghian ciru do hien tgi van chua dgt hieu qua tuong duong vdi cdc lodi khde. Mgt trong nhirng nguyen nhdn cua han che nay la su thanh thyc chua ddy dii ciia cdc trirng Ign nudi in vitro.
Da cd rat nhieu nghian ciru nham ndng cao chat lugng trung Ign thdnh thuc in vitro.
Leibfried vd dong tdc gid da nghian ciiu dya vdo trgng thdi ben ngodi ciia trirng nhu dp ddy cua Idp ta bdo can noan bao quanh noan bao trung dl chgn ra trirng cd dii chat lugng nudi thdnh thuc [8]. Trung cd ldp te bdo cgn noan day, toi thi ty lp thdnh thyc tdt hon [10].
Trirng dugc nudi trong mdi trudng cd bd sung them hormone FSH cd ty le d giai doan Mil
Tel 0973 113 541 Emad iigm'elinaimaiid-,f;niail a
cao hon so vdi khi dugc nuoi trong mdi trudng khdng cd FSH (lan lugt la 86,7 va 58,0%) [4], Miia vu ciing cd dnh hudng tdi chdt lugng trung Ign, khi thu trirng d miia xuan cd ty le thdnh thuc thap hon khi trirng thu d miia he (Idn lugt la 58,0 vd 66.1%)).
Nhiau nghien cuu chi ra rang mdi trudng cd bd sung dich nang trung (pFF) se kich thich triing thdnh thuc [12]. Qian va ddng tac gid da khao sat anh hudng cua kich thudc nang dan chat lugng thanh thuc trong dng nghiem Kat qud cho thay trirng thu d nang tren 5inm cd chat lugng thdnh thuc vd kha nang phdt trian sau thy tinh dan giai dogn 6-8 ta bdo cao han ban so vdi trirng duoc thu d nang 2-2,9mm (90,5% vdi 67,4%) va (34,5% vdi 7.7%) [12], 0 Viet Nam, viec thi nghipm nuoi thu tinh dng nghiem (TTON) trirng Ian da dugc tien hdnh dau nhirng nam 2000 [3]. Tuy nhien ty lp trirng thdnh thuc vd hipu suat tao phdi van cdn thdp (ty lp thanh thuc 14,8-18,3% [2]). Vi vay viec nghian cuu cdc yeu td anh hu'dng tii' dd ndng cao sy thdnh thuc ciia trii'ng cung nhu hieu suat TTON va tao phdi d ddi tugng Ign Id can thiat nham cd duac mot he thdng sdn 149
Nguyen VSn Ldm vo Dtg Tgp chl KHOA HQC & C 6 N G NGHE 12(12)/2: 149-154 xudt phdi hipu qua vd on djnh. Trong nghian
ciru nay chung tdi tim hiiu anh hudng cua kich thudc nang den chat lugng trirng Ign thdnh thyc in vitro.
VAT LI$U, NQl DUNG v A PHU'ONG PHAP NGHIEN c u t )
Vgt lipu nghien cuu
Trirng Ign thu tir buong trdng dugc lay tir Id md tran dja bdn thanh phd Ha Ngi, Ngi dung nghien cuu
Tren buong trung Ign chimg tdi chia ra lam ba nhdm nang cd kich thudc khac nhau; dudi 2mm; tir 2-3mm va tren 3mm.
Ngi dung I: Khao sat ty lp cdc nhdm nang cd mgt tren budng triing
Ngi dung 2: Ddnh gia anh hudng ciia kich thudc nang trung tdi chdt lugng trimg mdi thu.
Ngi dung 3: Ddnh gia kha nang thdnh thyc ciia trimg thu d cac nhdm nang sau 44 gid nudi.
Phuong phap nghien cuu
Thu vd phdn logi trung: Budng trirng Ign sau khi thu d Id md dugc bao qudn trong nhiet do tir 35-37 °C vd van chuyen ngay va phdng thi nghiem, Tai day budng trung dugc Idm sgch vd rua lgi nhieu lan trong dung djch NaCl 9%o trudc khi tiln hdnh thu trirng, 0 thi nghipm ndy chung tdi tiln thu trung bdng phuong phdp hiit nang, dimg xi lanh 10ml gdn vdi kim tiem 20G choc vao nang trirng vd hut phdn djch nang cd chira trirng. Trirng dugc thu bang kim thuy tinh cd kich thudc thich hgp vd riia lai 3-4 lan trong mdi trudng thu trirng vd phdn logi triing theo phuong phdp ciia Leibfried vd First, 1979 [8]. Tdt cd cdc
giai dogn dugc thuc hien trong dieu kien vo triing va d 3 7 ^ .
Nuoi thdnh thyc in vitro: Trung sau khi dugc phan logi, danh gia chdt lugng se dugc dtra vdo mdi trudng nudi thanh thyc theo phuong phap dugc md ta trong nghien ciru ciJa Kikuchi va dong tdc gia, 2002 [6], [7]. Trong thi nghipm nay chiing toi chi nudi va danh gia chat lugng thanh thyc cac triimg logi A va B.
Tirng nhdm 40-50 trung dugc nudi trong 0,5ml mdi trudng MAT-I (mdi trudng NCSU- 37 cd bo sung cdc hormone PMSG va hCG, cimg vdi dbcAMP) vd trong dTa nudi te bao4 gieng, trong tii dm 38,5°C va 5% CO2, dp 'm 95%. Sau 24 gid trong mdi trudng MAT-I, trirng dugc chuyin sang mdi trudng MAT-II (mdi trudng NCSU-37 khdng bl sung hormone vd dbcAMP) nudi tiep trong 20 gicr, Sau dd trirng dugc tach sach t l bao can noan vd danh gia ty Ie thdnh thyc.
Ddnh gid su thanh thuc cita trung: Dugc lam theo phuong phdp ciia Leibfried va Frist, 1979 [8]. Trung Ign sau khi nudi tiianh tiiuc dirpc tdch sach Idp tl bdo can noan bdng each xir ly trong gigt men hyaluronidase vd vi thao tic bdng cdc kim thuy tinh cd dudng kinh dau kim bdng vdi dudng kinh cua trimg. Sau dd nia lai 3 - 4 lan trong mdi tnrdng nudi thanh thyc (MAT- II), quan sat va ghi nhan sy cd mgt hodc khong cd mat cua thi cyc thu nhdt dudi kinh hien vi vdi dg phdng dai 40-100 lan.
KET QUA VA T H A O LUAN
Khdo sdt ty I? cdc nhom nang tren buong (rung Ket qud khao sat ty Ie cdc nhdm nang tren budng trirng dugc trinh bay trong bang I.
Bang 1. Kel cjud ty l^ cdc nhom nang co mgt tren buong trimg.
So buong (rung
31 TBso nang/BT
So nang trung 1983 64±5.4
Du'6'i 2mm I205(60,8±3,l)'
38,9 '±5,9
Nh6m nang triing Til' 2-3mm 518(26,1+2,0)'
16,7'±2,0
Tren 3mm 260(13,112,5)' -
8,4= ±2,1 Thi nghi4m duac lap lai 6 Idn. Ky hieu a. b. c trin ciing mdi hdng thi hiin su sai khde cd y nghia ihdng^
(P<O.OS)
NguySn V3n Lam vd Dtg Tiff chi KHOA HOC & CONG NGHE 112(12)/2: 149- 154
Kich rhu&c nang (mm)
Dual 2 Til 2-3 Tren 3
S6 trti-ng thu dtr(fc 825 860 626
B^ng 2. Kel qud phdn
Triing A (% ± SEM) 0(0)- 50 (5,8±2,3)' 128 (20,5±6,8)'
loai chat Itr^ng trirng Phiin loat chit Itfitng trthig
Triing B (% ± SEM) Triing C (% ± SEM) 334 (40,5±4,0)' 491 (59,5±4,7)"
543(63,1±4,9)' 267 (31,1±5,3)"
391 (62,5±7,8)' 107 (17,l±l,9)' Thi nghiem duac Igp lgi 7 Idn. Ki hiqu a, b, c tren ciing mdt cgt the hien su sai khde cd y nghTa thdng ke (P<0,05)
Tdng sd nang ciia 31 budng triing la 1983 nang, trung binh sd nang tren mdi buong tning khdo sat Id 64 nang/budng triing; nhdm nang cd kich thudc dudi 2mm cd trung binh nang/budng trii'ng cao nhdt 38,9 chiim 60,8%;
nhdm nang cd kich thudc tran 3mm cd trung binh nang/budng trirng thdp nhdt Id 8,4 chi chiim 13,1%) vd nhdm tir 2-3mm cd trung binh nang/budng trung Id 16,7 chiem 26,1%.
Kit qud nay khd phii hgp vdi kit qud khao sat ciia Ratky va ddng tdc gia [10] khi khao sat 60 buong trirng lgn ket qua thu dugc, sd nang tren bl mat mdi budng trirng dgt tir 60-90 nang, trong do sd nang cd kich thudc >2 mm Id 24,8 nang/budng triing. Nhu vay cd tha thdy khong cd sy sai khac ve sd lugng nang trimg -vd ty la cdc nang vdi kich thudc khac nhau giira nghien cuu ciia chiing tdi vdi nghien ciiu cua Ratky va ddng tac gid [10], ve cdc chi tieu tran cua cimg mdt gidng lgn d hai dja dilm nghian cuu cdch xa nhau vt dia ly cung nhu khi hau.
Anh hudng ciia kich thuac nang trung den chdt luang trung mai thu: Theo tieu chudn phan loai triing ciia Leibfied vd cgng sy (1979) [8]. Trirng A cd tir 3 ldp tl bao can noan trd len, bdng toi bao phu ddng deu tren mat te bao trirng vd noan bao chat cd dd ddng nhat cao vl mau sdc; Trung B cd tir 2 den 3 Idp te bao can noan, do bao phii ciia cac te bao can noan va miic dp dong nhdt mau sac ciia noan bdo chdt khdng bdng trirng A, trung C Id trirng cd hoac khdng cd ldp te bdo can
noan, noan bao chdt khdng ddng nhdt, Ung dung phuong phap phdn logi cua Leibfied vd cgng sy (1979) [8], chiing tdi thu dugc kit qud chi tiet dugc the hipn d bdng 2.
Trii'ng logi A d nhdm nang tren 3mm cd ty lp cao hon han so vdi 2 nhom con lai tir 2-3mm va dudi 2mm Idn lugt la (20,5±6.8%; 5,8±2,3 vd 0%). Trirng loai B d nhdm nang tran 3mm vd til' 2-3mm cd ty le tuang duong nhau (62,5±7,8% vd 63,1±4,9%). Trung loai C dugc thu d nhdm nang cd kich thudc dudi 2mm cd ty lp cao hon han so vdi nhdm nang cd kich thudc tir 2-3mm vd tren 3mm lan lugl la (59,5±4,7%; 31,1±5,3% va 17,1±1,9%) NhQ'ng kit qua tren cho thay kich thudc nang cd anh hudng ro ret tdi chit lugng trung lgn.
So vdi kit qua ciia tac gid Nguyan Thi Udc vd cdng sy khi khdo sat tran 90 buong triing lgn thu dugc ket qud sd lup-ng triing loai A vd B tir nang tren 2 mm Id 79,83 ± 0,03% [4], thi kit qud cua chung tdi thdp han khdng dang ke (74,8%i). Dieu do cho thdy sy dn dinh cua he thong khi trung duoc thu nhdn va nudi trong ciing miia vy.
Khd ndng thdnh thuc ciia trimg thu fir cdc nhom nang sau 44 gia nudl in vitro. Sau khi thu va phan loai trung, tien hdnh nuoi cdy triing trong mdi tru'dng MAT-I (mdi trudng NCSU-37 cd bd sung cac hormone PMSG va hCG, ciing vdi dbcAMP), trong dia nudi te bao 4 gieng, trong tii am 38,5''C va 5% CO2, do dm 95%, Kat qua dugc trinh bay cu tha d bang 3,
151
Nguyin Vdn Lam vd Dtg Tgp chl KHOA HQC & CONG NGH$ 1 l2(l2)/2: 149 -154 Bang 3.
Kich thudc nang Dudi 2 mm Tir 2-3 mm Tren 3 mm
Ddnh gid • thdnh ihyc bdng phuang phdp S6 triing
thf nghiam 192 175 150
soi Si
the cue sau 44 gia nudi
tm-ng th^nh thgc % I 3 ( 6 , 8 ± 3 , l ) ' 52 (29,7±4,6)' 91 (60,8±530.4)'
Thi nghi\'m ldp lgi 6 tdn. Ki hi^u a. h. c trin ciing mgl cgt the hii^'n .su khde nhau cdy nghTa vdi P<0.05 Kich thudc nang cd dnh hudng tdi chat lirgng Triing lgn thu dugc tir nang trung cd kich va t>' lp thdnh thyc ciia trirng Ign nudi trong thudc tren 3mm cd ty le thanh thyc cao nhat la dng nghipm, Ty lp thdnh thyc ciia trirng d 60,8%i khi nudi in vitro. Chiing tdi de nghi sii' nhdm nang cd kich thudc iron 3mm (60,8%) dyng cdc trung thu tir nang trimg cd kich thuoc cao hon hai nhdm cdn Igi. Tuy nhian so vdi tren 3mm de nang cao hieu sudt tao phdi.
mg so nghien ciru tran the gidi van cdn thap han rat nhieu (86.7% [5] va 90.5% [12]), Sy kem thdnh thyc khi nudi triing in vUro cd the do rat nhiau nguyan nhdn, trong dd cd dieu kien chan nudi d nude ta vdi che dg dinh dirdng chua hgp ly, thuc dn chira nhieu chdl kich thich tang trgng, vd dd tudi giet mo sdm hon so vdi do tudi trudng thdnh ciia con vat Nhu vay can cd nhii'ng nghian ciiu sdu hon nhdm cdi tien he thdng IVM cho triing Ign thu tai Viet Nam phuc vu cho tgo phdi bdng thy tinh dng nghiem {in vitro fertilization), kich thich mau sinh (parthenogenesis) hodc nhan ban vd tinh (cloning).
KET LUAN
- Qua khdo sat chiing tdi riit ra kit ludn: trOrng lan thu dugc tii' nhdm nang cd kich thudc dudi 2mm chiem ty le 60,8%.. nhdm nang cd kich thudc tren 3mm chi chiim ty le 13,1%
\anhdm tir 2-3mm chiim ty le Id 26,1%.
- Trung Ign logi A d nhdm nang trung cd kich thudc tren 3mm cd ty le cao nhdt (20.5±6,8%); Trirng Ign loai B d nhdm nang trirng cd kich thudc tren 3mm vd tir 2-3mm cd ty le tirong dirong nhau (62,5±7.8% va 63,1±4,9%). Trirng logi C d nhdm nang trirng CO kich thudc dudi 2mm cd ty le cao nhdt (59.5±4,7%). Kit qud tren cho thay kich thudc nang trirng cd dnh hudng rd ret tdi chdt lugng trirng Ign.
152
Hinh 1 Tdp hgp te hdo trirng thu dugc vd phdn logi trirng ng A. B. tnmg B: C: trirng C)
Hinh 2. Trung xudl hiin thi cue thu nhdl
Nguyen Van Ldm vo Dig Tgp chi KHOA HOC & CONG NGHE ll2(12)/2: 149- 154
T A I LIEU T H A M K H A O [1]. Nguyin Thj Phucmg Hiln (2007), Nghiin cuu anh hudng ciia miia vy lin ket qud nudl thanh thuc mgl sd dgng vdt nudi. Lugn vdn thac sy Nong nghiep.
[2]. Huynh Thj Le Duyen, Phan Kim Nggc, H i Huynh Thdy Ducmg, Nguyen Q u i c Dgt (2003),
"ijng dyng ky thugt thy tinh trong ong nghifm tren heo", Hgt nghi cdng ngh4 sinh hgc todn qudc.
Hd Ngi, tt.639-642.
[3]. Bill Xudn Nguyen (2003), "Phdi iriin cdng ngh4 phdi vd ti bdo phoi d Vi4t Nam ", Ky yeu Vien C6ng nghp sinh hpc. Nxb Khoa hpc vd Ky thugt. Ha Npi, tr. 411 - 4 1 7
[4]. Nguyen Thi Udc, Nguyin Viet Linh. Nguyen van Hgnh, Qudn Xuan HChi, Ddng Nguyin Quang Th^nh, Trdn Thj Tham, Nguyen Thj Min, BCii Linh Chi, Nguyen Trung Thdnh, Duong Dinh Long, Nguyin Khde Tich. Phan Ngpc Minh, BCii Xudn Nguyen (2008), "Nghien ciru san xuat phoi Ign mini npi dja bang t l hgp cdng nghp Ing nghifm vd nhdn bdn vo tinh ", Tap chi cdng nghi sinh hgc, 6{4A),tr. 625-635.
[5]. Schoevers E,J., Kidson A., Verhijden J.H.M., Bevers M,M. (2003), "Effect of follicle- stimulating hormone on nuclear and cytoplasmic maturation of sow oocytes in vitro".
Theriogenology, 59: 2017-2028,
[6]. Kikuchi K., Onishi A., Kashiwasaki N., Iwamoto M., Noguchl J., Kaneko H., Akita T., Nagai T. (2002), "Successful piglet production al^er transfer of blastocyst produced by a modified invitro system", Biol Reprod., 66: 1033-1041.
[7], Kikuchi K., Kashiwazaki N., Noguchi J„
Shimada A., Takahashi R , Hirabayashi M , Shino M„ Ueda M., Kaneko H. (1999),
"Developmental competence, after transfer to
recipients, of porcine matured, fertilized, and cultured in vitro". Biol Reprod., 6:336-340.
[8]. Leibfried L. and First N,L. (1979),
"Characterization of bovine follocular oocytes and their ability invitro". J. Anim. 5c/.,48:76-86.
[9]. Ni Wayan Kurniani Karja. (2008), "Nuclear Maturation of Porcine Oocytes in vitro: Effect ofthe Cumulus-Oocyte Complexes Quality", Indonesian Journal of Biotechnology., 13 (2), 1078-1084.
[10]. Ratky. Katiyar P K., Raminder Singh.
(2003), "Maturation of porcine oocytes in vitro and developmental competence of porcine oocytes", Biology of reproduction , 1 1 : 299-305.
[ I I ] . Tatemoto H., Sakurai N , Muto N,( 2000),
"Protection of porcine oocytes against apoptoic cell death caused by oxidative stress during In vitro maturation: role of cumulus cells". Biol Reprod.63:%Q5-%\Q.
[12], Yun Qian,, Wei Qun Shi,, Jia Tong Ding., Bi Qin Fan., Yutaka Fuku. (2001), "Effect of follicle size on Cumulus - Expansion, In vitro fertilization and development of porcine follicular oocytes", Journal of Reproduction and Development., 47(3), 1 4 5 - 1 5 2 .
Ldi cdm on: Cdc tdc gid xin chdn thdnh cdm an TS. Bui Xudn Nguyen. TS. Nguyen Thi Vac vd cdc dong nghiep tai Phong Cong nghe Phoi, Vien Cong nghe sinh hoc dd giiip da thuc hien vd dong gop y kien. Nghien cuu duac tdi tra bdt de tdi NAFOSTED 106.I6.J4L09, chii nhiem di tdi TS. Bid Xudn Nguyen.
Nguyin van Ldm vd Dtg Tap chi KHOA HQC & C 6 N G N G H $ 112(l2)/2: 149 -15<)
SUMMARY
EFFECTS OF FOLLICLE SIZE ON OOCYTE QUALITY AFTER IN VITRO MATURATION
Nguyen Van Lam', Hua Nguyet Mai*', Nguyen Van Hanh', Nguyen Viet Linh' ' Insiiiule of Biotechnology - Vietnam Academy of Science and Technology
'College of Science -TNV In this experiment, uc examined the proportion of groups of different sized follicles on the surface of pig ovaries and evaluate the effects of follicle size to quality oocytes during in v//ra culture On ovaries, we are divided into 3 groups of different sized follicles under 2mm, 2-3mm and >3mm the results is (60.8 ± 3,1% and 26,1 ± 2.0% and 13.1 ± 2.5%, respectively) (P <0.05). The rate of grade A oocytes in groups of 3 mm follicles than group 2-3mm follicles (20.5 ± 6.8% and 5.8 ± 2.3%). while less than 2 mm without the oocyte capsule type A (0 %) (P <0.05). Oocytes grade B from group 2-3mm and >3mm similar rate (63.1 ± 4.9% and 62,5 ± 7.8%) is less than 2 mm follicles in the lower group was only 40,5 ± 4.1% (P <0.05). The oocytes in the group qualify conducted follicles cultured in 38.5"C and 5% CO2 and 95% N. The results showed that the rate of oocyte maturation in the highest group 3mm is 60.7 ± 5,4% followed by group 2-3mm follicles for maturation rate is 29.7 ± 4,6% and less than 2 mm follicle group is 6.8 ± 3.1% (P <0.05). Follicle size affects the results obtained and the rate of oocyte maturation in vitro culture process. The follicles size >3 mm for best results both in terms of good oocytes and oocyte maturation rate after the in vitro maturation.
Key words' Follicle size, Porcine oocyte. Quality, Maturation
Phdn bien khoa hoc: TS. Nguyin Thi Hdi Yin - Trudng Dgi hoc Khoa hgc - DH Thdi Nguyen ' Tel 0973 113 541. Emad. nsuvetmaimaiid'emad r,
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