研究旨在根据基因多态性和超数排卵效果的关联分析,找到一种能预测水牛超数排卵反应的因子.以127头水牛(摩拉、尼里、地中海、本地、杂交水牛)为材料,采集血样,提取基因组DNA.采用PCR及DNA测序技术,以GenBank公布的牛FSHR (follicle stimulating hormone receptor)、INHA(inhibin alpha)、LHCGR(luteinizing hormone/choriogonadotropin receptor)、OPN (osteopontin)基因序列为参考序列,设计PCR引物,对FSHR的5’上游调控区、INHA第1外显子、LHCGR第11外显子和OPN3907启动子进行了基因型检测,并将基因多态性与45头水牛的超数排卵性状进行了关联分析.结果表明:FSHR、INHA、LHCGR的相应SNP位点都不存在多态性;OPN基因启动子上游存在T碱基插入/缺失突变,T9/T9、T10/T10、T9/T10的基因型频率分别为:0.4020、0.1275和0.4634.此外,T10/T10型水牛在第5次超数排卵时获得的可用卵母细胞数显著高于T9/T9型水牛(P＜0.05).以上结果预示,OPN可作为水牛超数排卵效果的一个预测因子.

为了筛查河流型水牛种公牛染色体罗伯逊易位,试验采用常规微量全血培养和空气干燥制片法对摩拉水牛、尼里/拉菲水牛、地中海水牛及其杂交后代共49头河流型水牛种公牛进行染色体的遗传检测和分析.结果表明:河流型水牛种公牛染色体数目均为2n=48+ XY,包含5对双臂(中部、亚中部或亚端部)常染色体、19对端部着丝粒常染色体和1对性染色体,未发现染色体罗伯逊易位现象.

This study was conducted to investigate the effect of different linolenic acid ( ALA) concentrations ( 0, 10, 50, 100 and 200μmol· L-1) during in vitro oocyte maturation (IVM) and in vitro embryo culture (IVC) on the maturation of oocytes and developmental competence of preimplantation embryo in buffalo.The oocytes from abattoir ovaries were matured and fertilized, and presumptive zygotes were cultured in the medium, then the maturation rate cleavage and blastocyst development rate were examined, respectively.The results showed that the maturation rate of oocytes treated with 50 μmol· L-1 ALA was significantly increased ( 74.18%) compared with the control and other supplemental groups ( P<0.05 ) , and the blastocyst development rate of oocytes treated with 50 μmol· L-1 ALA (33.24%) was significantly higher than those of the control and 10 μmol· L-1 ALA supplemental groups ( P <0.05).The presence of 50 μmol· L-1 ALA in IVC resulted in a significantly higher blastocyst development rate (34.52%) compared to the control and 100μmol· L-1 ALA supplemental groups (P<0.05);The ALA with differ-ent concentrations were added to both IVM and IVC, cleavage rates in the group of 200 μmol· L-1 ALA was signifi-cantly higher than that of the control (P<0.05), while blastocyst development rate and percentage of Day 7 blasto-cysts showed no difference.In conclusion, addition of 50 μmol· L-1 ALA to either IVM or IVC in vitro can promote the maturation rate of buffalo oocytes and blastocyst development.

水牛适应和抗病力性强、耐高温高湿、耐粗饲、易饲养、使用年限长,非常适合我国南方的饲料资源和气候条件,而且水牛奶营养丰富,现今我国正致力于发展水牛奶业.引入的河流型水牛,体格大,生长快速,产奶量高,与本地水牛杂交后代的奶水牛产奶量达到1200～2000kg,可显著提高本地水牛的产奶性能.但由于水牛的繁殖性能低下,我国水牛杂交改良面临着良种种源匮乏的问题,要突破这个瓶颈,必须集成现代动物繁殖技术(如人工授精、超数排卵、体外胚胎生产、核移植、转基因和胚胎移植等技术),以提高水牛的繁殖效率和种源供应能力,从而促进我国水牛品种改良和水牛奶业开发.近年来,围绕水牛繁殖生物技术已开展了大量的研究,下面就此作一综述.

This study was conducted to optimize the culture and cryopreservation conditions for buffalo hand-made clone (HMC) embryos. Microdrop (MD), well of the well (WOW), flat surface (FS) systems were adopted to culture HMC reconstructed embryos in vitro. The derived HMC blastocysts were vitrified either with 40% EG, 25% EG + 25% DMSO or 20% EG + 20% DMSO + 0.5 M sucrose. Furthermore, the efficiencies of HMC and traditional somatic cell nuclear transfer (SCNT) were also compared. The results showed that: (1) the cleavage rate of HMC embryos cultured in WOW was higher than those cultured on FS (70.5% vs 55.8%, p<0.05) and in MD (70.5% vs 50.0%, p<0.01); also, the blastocyst rate of HMC embryos derived from WOW system (40.0%) was higher than those derived from FS (19.8%) and MD (8.3%) systems (p<0.01); (2) the cryosurvival rate of blastocysts vitrified with 20% EG + 20% DMSO + 0.5 M sucrose was higher than those vitrified with 40% EG (90.9% vs 62.0%, p<0.01); (3) both the fusion and blastocyst rates of HMC reconstructed embryos were higher than those of SCNT reconstructed embryos (89.1% vs 79.8%, p<0.05; 40.0% vs 19.8%, p<0.01), whereas the cryotolerances of HMC and SCNT blastocysts were not different from each other (90.9% vs 92.3%). In conclusion, WOW is the most suitable method for culture of HMC embryos, and vitrification of HMC blastocysts with 20% EG + 20% DMSO + 0.5M sucrose results in a high cryosurvival rate. HMC can be an alternative to traditional SCNT in buffalo.

Transgenic somatic cell nuclear transfer (SCNT) is currently one of effective ways for producing transgenic animals, but the efficiency of the exogenous gene integrating into somatic cells is still low. To obtain stably transfected buffalo somatic cells for transgenic SCNT for further experiments, in this study, buffalo cumulus cells were transfected with pEGFP-N1 by using Lipofectamine 2000 and selected by exposing to G418 (600 μg/ml) for 3 weeks. Selected transfected cells were imaged under a fluorescence microscope. After 24 hours following transfection, approximate 5% of cells were transiently transfected. Following G418 treatment for 3 weeks, 15% of the survived cells expressed EGFP with fluorescence intensity several folds larger than that in the cells without G418 selection (control). EGFP expressing cells, even part of non-EGFP expressing cells with G418 selection (treatment) were detected to have complex shapes in their morphology which is different from the typical fusiform shape of cumulus cells (control). This study demonstrates that EGFP can successfully be expressed in buffalo cumulus cells and stably transfected cells can be enriched by G418 selection for multiple purposes. Future study should be done to improve the integration rate of exogenous gene and the percentage of stably transfected cells.

为了探讨屠宰场卵巢体外生产胚胎(IVF)、OPU(ovum pick-up)-IVF胚胎以及体细胞核移植胚胎(SCNT)的发育能力和发育速度,研究比较了3种不同来源胚胎的卵裂率、囊胚率和囊胚形成时间。结果:屠宰场卵巢-IVF、OPU-IVF和SCNT胚胎的分裂率没有显著差异(52.5%、63.4%、56.4%,P>0.05),而OPU-IVF胚胎的囊胚发育率显著高于SCNT胚胎(36.4%vs 19.8%,P<0.05);对屠宰场卵巢-IVF胚胎而言,7 d囊胚的比率(46.8%)高于6 d、8 d和9 d的囊胚比率(17.7%、23.0%、12.5%,P<0.01);OPU-IVF 7 d囊胚的比率(44.5%)高于6 d、8 d和9 d的囊胚比率(17.6%、27.8%、9.7%,P<0.01)。SCNT 6 d和7 d的囊胚比率(49.6%、31.3%)高于5 d、8 d和9 d的囊胚比率(7.4%、8.5%、1.7%,P<0.01)。试验表明SCNT胚胎的发育能力要显著低于OPU-IVF胚胎,而OPU-IVF胚胎与屠宰场卵巢-IVF胚胎的发育能力没有显著差异;SCNT胚胎的囊胚形成时间早于IVF胚胎。

The present study investigated the effects of curcumin on buffalo embryonic development by incubating zygotes without or with the compound during in vitro culture (IVC). Cumulus-oocyte complexes retrieved from antral follicles of water buffalo ovaries were matured for 20-22 hrs and inseminated with motile buffalo sperm in Tyrode's medium for 24 hrs. The presumed zygotes were washed 3 times and transferred into 50 mu l droplets of IVC medium (TCM 199 + 10% FBS) supplemented with curcumin in various concentrations (0 mu M, 2.5 mu M, 5 mu M, 10 mu M and 20 mu M, respectively) and co-cultured with buffalo cumulus cells monolayer for more than 7 days to evaluate the developmental ability of embryos (0, 2.5, 5, 10 and 20 mu M groups, respectively). The cleavage rate (CR) and blastocyst rate (BR) were assessed at 48 hrs and 168 hrs, respectively, after fertilization (0 hrs). The results showed that there was a slight decrease in the CRs from 0 mu M group (control) to 20 mu M group (60.34%, 56.18%, 57.69%, 55.09% and 45.58%, respectively) but there was no statistical difference among all groups. However, the BR in 20 mu M group (9.82%) was significantly lower (p<0.05) than those from 0 mu M to 10 mu M groups (30.61%, 26.30%, 26.80% and 27.48%, respectively). Thus, we concluded that exposure of buffalo zygotes with high-dose curcumin (20 mu M) during IVC has injurious effects on embryonic development in vitro.

This study was conducted to investigate the developmental competence and blastocyst formation speed of three kinds of in vitro produced buffalo embryos: in vitro fertilized (IVF) embryos derived from oocytes collected either by aspirating of abattoir ovaries (abattoir-IVF) or by ovum pick up (OPU-IVF) matured in vitro, and somatic cell nuclear transferred (SCNT) embryos. The cleavage rate, blastocyst rate and blastocyst formation time of these embryos were recorded. The results showed that: (1) the cleavage rates of abattoir-IVF, OPU-IVF and SCNT embryos were not different form each other (52.5%, 63.4%, 56.4%, respectively), whereas the blastocyst rate of OPU-IVF embryos was higher than that of SCNT embryos (36.4% vs 19.8%, p<0.05); (2) as for abattoir ovaries-IVF embryos, the percentage of blastocysts harvested on Day 7 (46.8%) was higher than those harvested on Days 6, 8 and 9 (17.7%, 23.0%, 12.5%, respectively; p<0.01); as for OPU-IVF embryos, the percentage of blastocysts harvested on Day 7 (44.5%) was higher than those harvested on Days 6, 8 and 9 (17.6%, 27.8%, 9.7%, respectively; p<0.01); as for SCNT embryos, the percentages of blastocysts harvested on Day 6 and Day 7 (49.6% and 31.3%) was higher than those harvested on Days 5, 8 and 9 (7.4%, 8.5%, 1.7%, respectively; p<0.01). In conclusion, the developmental competence of SCNT embryos was poorer than that of OPU-IVF embryos, whereas the abattoir-IVF and OPU-IVF groups showed similar developmental competence. Furthermore, the blastocyst formation of SCNT embryos occurs earlier than the IVF embryos.

In the present study, chromosome specimens were prepared by culture of peripheral blood lymphocytes, and karyotypes were analyzed routinely from different varieties of Mediterranean buffalo (M), F1 of Mediterranean buffalo x Murrah buffalo (MM), F1 of Mediterranean buffalo x Nili-Ravi buffalo (MN) and F1 of Mediterranean buffalox Guangxi native swamp buffalo (MG). The results were shown that the chromosomes of M, MM and MN are 2n=50, which are in consistence with those of river buffaloes of Murrah and Nili-Ravi reported by other researchers. However, the chromosomes of MG are 2n = 49, which are disagree with 2n=48 of swamp buffalo and 2n=50 of riverine buffalo. These results confirmed that Mediterranean buffalo belongs to the riverine subspecies as the same as Murrah and Nili-Ravi instead of the swamp subspecies as the Guangxi native buffalo.

The present study investigated the effect of linolenic acid supplementation on the early development of buffalo embryos in vitro. The presumptive zygotes were transferred to the vitro culture medium (TCM199, 10% FBS) supplemented with 0 (control), 10, 50, 100 and 200 mu M linolenic acid. Cleavage and blastocyst development rates were recorded on Day 2 and Day 6 to 9 after insemination, respectively. A total of 1482 COCs were used in ten independent replicates. The results showed that the cleavage rates in the groups of 0 (control), 10, 50, 100 and 200 mu M linolenic acid were 59.0 +/- 7.2%, 62.7 +/- 6.3%, 63.5 +/- 6.1%, 58.1 +/- 7.9% and 60.7 +/- 6.7%, respectively. No statistical difference was observed regarding cleavage rates among treatments (p>0.05). The treatment of COCs with 50 mu M linolenic acid resulted in a significantly higher percentage of blastocyst development rate compared to the control group and those supplemental with 10, 100 or 200 mu M linolenic acid (34.5 +/- 4.1% vs 25.4 +/- 8.2%, 28.85 +/- 7.59%, 26.41 +/- 9.51%, 29.45 +/- 10.60%, respectively, p<0.05). In contrast, no significant differences were found between the control group and the other treatments. In conclusion, the results of this study indicated that supplementation of linolenic acid in the vitro culture medium could enhance the blastocyst development in buffalo species and the optimal concentration of linolenic acid in the present procedures was 50 mu M.

以水牛rRNA基因的间隔序列为靶位点,构建多位点基因打靶载体,为建立转Fat1基因水牛研究获得关键材料.采用LA-PCR技术克隆了水牛rRNA基因家族序列,经常规PCR和DNA测序验证,成功构建了水牛多位点基因打靶载体.本研究建立的多位点基因打靶技术将部分解决目前存在的打靶效率低、安全性差等问题,为转基因水牛的研究奠定坚实的基础.

This study was conducted to optimize the culture and cryopreservation conditions for buffalo hand-made clone(HMC) embryos.Microdrops(MD),well of the wells(WOW),flat surface(FS) were adopted to culture HMC reconstructed embryos;and 40% EG,25% EG(ethylene glycol,EG) + 25% DMSO(dimethylsulphoxide,DMSO) and 20% EG + 20% DMSO + 0.5 mol/L sucrose were adopted to vitrify HMC blastocysts.Furthermore,the efficiency of HMC and traditional somatic cell nuclear transfer(SCNT) were also compared.The results showed that the cleavage rate of embryos cultured in WOW was higher than those cultured on FS(P0.05) and in MD(P0.01);the blastocyst rate of embryos derived from WOW system(40.0%) was also higher than those derived from FS(19.8%) and MD(8.3%) systems(P0.01);the cryosurvival rate of blastocysts vitrified with 20% EG + 20% DMSO + 0.5 mol/L sucrose was higher than those vitrified with 40% EG(P0.01);both the fusion rate and blasotycst rate of HMC reconstructed embryos were higher than those of SCNT reconstructed embryos(P0.05;P0.01),while the cryotolerances of HMC and SCNT blastocysts were not different from each other.These results indicated that HMC could be an alternative to traditional SCNT in buffalo,WOW was most suitable for culture of HMC embryos,and vitrification of HMC blastocysts with 20% EG+20% DMSO+0.5 mol/L sucrose could get a reasonable cryosurvival rate.

The effects of injecting timing after fertilization(5 to 6,9 to 10,13 to 14,17 to 18,21 to 22 h) and exogenous DNA concentrations(10,50 and 100 μg/mL) on the development of Fat-1 transgenic embryo were investigated.The results showed that the EGFP expressing rate and blastocysts'EGFP expressing rate of 9 to 10 h group were the highest,the EGFP expressing rate were significantly higher than 5 to 6 h group(P<0.01) and the blastocysts'EGFP expressing rate significantly higher than 21 to 22 h group(P<0.01);in the concentration test,the EGFP expressing rate and blastocysts'EGFP expressing rate of 50 μg/mL group were the highest,and significantly higher than 100 μg/mL(P<0.01).

Days 6~7 buffalo SCNT and the IVF blastocysts derived from abattoir ovaries and OPU were cryopreserved by slow freezing and vitrification in different cryoprotectant mediums.The 40%EG,25%EG+25%DMSO and 20%EG+20%DMSO+0.5 mol/L sucrose were adopted for vitrification,while 10% glycerol and 0.05 M trehalose dehydrate+1.8%EG+0.4%BSA were used for slow freezing.The results showed that(1): the efficiency of different vitrification medium was different from each other,20%EG+ 20%DMSO+0.5 mol/L sucrose was the best.As for slow freezing,the efficiency of the two kinds of medium was not different from each other.(2): 76.5% of the slow frozen blastocysts survived in 0~24 h after thawing,which was higher than that of vitrified blastocysts(48.9%).On the contrary,more vitrified blastocysts(42.6%) survived in 24~48 h than the slow frozen blastocysts(23.5%).In conclusion,the efficiency of vittification is higher than slow freezing,and the medium of 20%EG+20%DMSO+0.5 mol/L sucrose is best,while the slow frozen blastocysts survived earlier than the vitrified ones after thawing.

【Objective】The objective of this study was to investigate the effect of cumulus cells on in vitro maturation（IVM）,in vitro fertilization（IVF） and embryo development in buffalo.【Method】Five groups were divided by the oocyte with cumulus or not to be cultured of in vitro maturation and fertilization,which were group I：natural denudate oocytes（DO）,group Ⅱ：natural DO ＋ cumulus cells,group Ⅲ：mechanical DO ＋ cumulus cells,group Ⅳ：Grade A and B cumulus oocyte complexes（COCs） removed cumulus cells before IVF,and group Ⅴ：Grade A and B COCs,respectively.Furthermore,the effects of bovine and buffalo granulosa cells cocultured with IVF-presumptive zygotes on the development of buffalo embryos were also compared.【Result】The results indicated that：group Ⅰshowed no difference with group Ⅱ（P0.05） in IVM rate,while it was significantly lower than group Ⅲ（P0.05） and group Ⅴ,Ⅳ（P0.01）;As for embryo cleavage rate,there were no difference between groups Ⅰ,Ⅱ and Ⅲ,while group Ⅰwas significantly lower than groups Ⅴand Ⅳ（P0.01）;For blastocyst rate,group Ⅴ was significantly higher than other groups（P0.01）.The blastocyst rate of buffalo IVF presumptive zygotes cocultured with buffalo granulosa cells was significantly higher than those cocultured with bovine granulosa cells（P0.05）.【Conclusion】Cumulus cells could promote the efficiency of IVM,IVF and embryo development in buffalo,and the buffalo granulosa cells monolayer had a much better effect on buffalo embryo development than bovine＇s when cocultured with buffalo zygotes.

The hand-made cloning(HMC) technique describes a simplified nuclear transfer process without the need for micromanipulators.This study was conducted to investigate the effect of different enucleation,fusion methods and donor cell types on the efficiency of HMC in buffalo.The results indicated that chemically assisted enucleation with cytochalasin b(CB) showed similar enucleation efficiency to Hoechst 33342 staining enucleation;the fusion rate of one-step method was significantly higher than two-steps method(P0.01),while the developmental abilities of the couplets reconstructed by the two methods were no significant different from each other(P0.05).The fusion rate of HMC couplets derived from ear fibroblasts was significantly lower than that of couplets from cumulus cells(P0.01),while the cleavage and blastocyst rates of the embryos from the two cell types were not different from each other(P0.05).The study showed that CB assisted enucleation,one-step fusion,cumulus cells can be adopted for HMC in buffalo to improve the efficiency of cloning.

To evaluate the influence of epidermal growth factor(EGF) on buffalo early embryo development and apoptosis in vitro,oocytes from buffalo ovaries at a slaughterhouse were matured and fertilized,and presumptive zygotes were cultured in the medium supplemented with EGF(0,25,50 or 100 ng/mL),then examined cleavage and blastocyst rate,respectively.After staining with Annexin V-Fluos Apoptosis Derection Kit(Annexin-V-Fluos Staining kit),apoptosis and necrosis of blastocyst under fluorescent microscope was examined.The result showed that the hatched rate in addition of 50 ng/mL EGF was significantly higher than that in controls(P0.05),and apoptosis rate and necrosis rate were significantly lower than those in controls(P0.05).While the cleavage rate,blastocyst rate,D7 blastocyst rate and the hatched rate in 100 ng/mL EGF in culture medium were lower than those in other groups,but the apoptosis rate and necrosis rate were higher.In conclusions,incubation of buffalo embryo in the presence of EGF improved the rate of hatched blastocyst and reduced the apoptosis of in vitro produced blastocysts.

This study was conducted to evaluate the effects of different hormone combinations with FSH, PGC, LHRH-A3 and LH on superovulation in river buffaloes. Thirty-five heads of river buffaloes were divided into six groups as follows: Group I, FSH (Japan, total doses 26 AU) + PGc (Shanghai, 0.6 mg); Group II, FSH (Japan, total doses 26 AU) + PGc (Shanghai, 0.6 mg) + LHRH-A3 (Made in Ningbo, 50 mu g); Group III, FSH (Beijing, total doses 20 mg) + PGc (Shanghai, 0.6 mg) + LHRH-A3 (Ningbo, 50 mu g); Group IV, FSH (Beijing, 20 mg) + PGc (Shanghai, 0.6 mg) + LH (Ningbo, 50 mu g); Group V, FSH (Canada, 800 mg) + PGc (Shanghai, 0.6 mg); Group VI, FSH (Canada, 800 mg) + PGc (Shanghai, 0.6 mg) + LHRH-A3 (Ningbo, 50 mu g). The results showed that superovulation rate was 97.14% (34/35). There were 8.71 mature follicles per head in superovulation (296/34). The average number of CL was 5.0 (170/34). The average ovulation rate was 57.3% (170/296). The average number of embryo collection was 2.72. Average transferable embryos were 1.33 (24/18). Recovery rate and transferable rate were 39.84% and 48.98 %, respectively. The mean number of CL in Group II (6.86 +/- 5.96) and the ovulation rate in Group VI (76.92 %) were the highest among the six groups. The results showed that the ovulation rate in Group VI with LHRH-A3 was higher than those in the other groups without LHRH-A3 (12.12%) and that with LH treatment (28.64%), respectively.

The objective of this study was to explore the effects of buffalo follicular fluid(BuFF) and bovine follicular fluid(BoFF) on the IVF of buffalo oocytes obtained from Ovum pick-up(OPU) and abattoir,in order to develop IVF system and improve embryo production efficiency in buffalo.According to the replacement amount of fetal bovine serum(FBS) by FF in IVM medium,oocytes were distributed randomly into 4 groups.group Ⅰ: 0% FF+ 10% FBS,group Ⅱ: 5% FF +5% FBS,group Ⅲ: 10% FF+ 0% FBS and group Ⅳ: 15% FF+ 0% FBS.As for addition of BuFF,the results showed that BuFF did not improve the cleavage rate of OPU and abattoir-derived oocytes after IVF(P0.05).However,addition of 5% and 10% BuFF have significantly improved the develop efficiency of IVF embryos,the blastocyst formation rates of groups Ⅱ and Ⅲ were significantly higher than those of groups Ⅰand Ⅳ(P0.01),and there was no difference between groups Ⅱ and Ⅲ(P0.05).The blastocyst formation rate of group Ⅳ was lower than that of group Ⅰ,but the difference between two groups were not significant(P0.05).As for the addition of BoFF,the cleavage rate and blastocyst formation rate of OPU-derived oocytes in group Ⅲ were significantly higher than those of groups Ⅰand Ⅳ(P0.01),and there was no significant difference between groupsⅠand Ⅱ(P0.05).BoFF did not improve the cleavage rate of abattoir-derived oocytes after IVF(P0.05).However,the blastocyst formation rate of groups Ⅱand Ⅲ were significantly higher than those of groups Ⅰ(P0.05) and Ⅳ(P0.01),and blastocyst formation rate of group Ⅳ was significantly lower than that of group Ⅰ(P0.05),while the blastocyst formation rate was significantly different between groups Ⅱand Ⅲ(P0.05).In conclusion,addition of 5% to 10% BuFF or BoFF in IVM medium in place of FBS can significantly improve the efficiency of IVF embryo production in buffalo,and BuFF shows a better effect.