奶牛胚胎分割试验研究

简单分割7～8天奶牛胚胎59枚(117个半胚)。裸半胚成对移给59头奶牛或黄牛,80～90天有27头(45.8％)妊娠。最后24头受体产犊34头,半胚产犊率29.1％(34/117)。有10对双胎,双胎率41.7％(10/24)。比较在不同情况下—7天或8天;晚桑椹或囊胚期;透明带软化处理或不软化处理—分割的半胚,成对移植后受体妊娠率分别是40.0％和57.9％;35.7％和54.8％;48.6％和41.7％。半胚产犊率分别是26.6％和34.2％;23.6％和33.9％;30.0％和27.7％。均无显著差异(P>0.05)。分割优质胚胎得到最好的(35.7％)半胚产犊率。半胚在体外5小时内移植有较高(30.5％)的产犊率。试验探索了奶牛半胚移给远处分散的农户黄牛的可能性。11对奶牛半胚移给11头黄牛有6头(54.5％)妊娠。最后5头受体产下7头奶牛犊,两对同卵双胎。     

奶牛冷冻胚胎分割和分割后半胚冷冻技术

用简易分割方法,分割奶牛冷冻胚胎16枚,获得半胚32枚,分割成功率为100％(16/16),可移植半胚29枚,移植于16头受体牛,到第3个情期未返情者经直肠检查有6头妊娠,妊娠率为37.5％(6/16)。其中,在分割前或者分割后经恢复培养0.5～2h再移植的10头受体牛,5头妊娠;而未经恢复培养,分割后直接移植的6头受体牛中,只有1头妊娠。移植后3个月,直肠检查确定2头流产。已有1头受体黄牛生出1头奶牛牛犊(母)和1头受体奶牛产1头奶牛牛犊。其余的2头妊娠受体牛将于9月份产犊。此外,用简易分割法分割奶牛胚胎5枚,得到半胚10枚,裸半胚直接冷冻,解冻后回收可移植半胚5枚,移植于4头受体牛,无一头妊娠。结果表明,冷冻胚胎的分割半胚优于分割后冷冻半胚移植效果;冷冻胚胎分割前或者分割后恢复培养移植优于未经恢复培养而直接移植;简易分割法可应用于冷冻胚胎的分割。     

奶牛冷冻胚胎分割移植效果探讨

分割加拿大奶牛冷冻胚胎 ,借荷斯坦牛受体移植。共分割冷冻胚胎 1 5枚 ,分割为裸半胚 30个 ,分割成功率 1 0 0 %。移植受体 2 7头 (其中移植一对半胚的受体 3头 ) ,移植妊娠 1 4头 ,受体移植妊娠率51 .85% ,产犊 1 4头 ,半胚产犊率为 4 6 .6 7% ,按未分割的整胚计算 ,产犊率达到 93.34 % ,比整胚移植产犊率还提高 1 8.34个百分点。移植半胚生产 1头犊牛需胚胎 1 .0 7枚 ,比整胚减少 0 .2 6枚 ,降低成本1 51 0 .6 0元。说明胚胎分割是降低成本 ,扩大卵源 ,增加良种牛数量的有效方法。     

奶牛新鲜和冷冻胚胎分割移植试验

用简单方法,分割7～8日龄新鲜牛胚胎(1分为2),裸半胚成对移植给66头受体,90天妊检,移植妊娠率为56.1％(37/66)。除6头流产和尚有5头待产外,已有26头受体产犊35头,其中有9对同卵双胎,双胎率为34.6％(9/26),半胚产犊率为29.2％(35/120)。对影响成对半胚移植妊娠率和半胚产犊率的诸多因素如胚胎质量,胚胎在体外停留时间、胚胎发育阶段、受体牛品种、黄体状况等进行了较系统的研究。同时对冷冻胚胎进行了分割试验,移植妊娠率为45.5％(5/11),已产3头犊牛。对快速冷冻和常规冷冻胚胎分割后的移植妊娠率进行了比较,分别为25.0％(1/4)和57.4％(4/7)。     

移植奶牛胚胎冷冻后分割成的二分胚生产同卵双生犊牛的实验

对解冻后品质优良的Ａ级囊胚进行分割，并将二分胚移植生产同卵双生犊牛。结果表明：９枚Ａ级囊胚切割成１８枚半胚，胚胎分割成功率１００％。单胚移植受体６头。妊娠２头，妊娠率３３．３％；双半胚移植６头，妊娠２头，妊娠率３３．３％。     

走近奶牛胚胎移植

一、何为奶牛胚胎移植 奶牛胚胎移植就是将一头优良品种的奶牛配种后的早期胚胎取出,移植到另一头母牛体内,使之继续发育。提供胚胎的母牛称为供体,接受胚胎的母牛称为受体。在自然繁殖中,一头高产奶牛一生最多生十几头后代,而胚胎移植可使其获得百余头后代。 国外的胚胎移植技术兴起于20世纪70年代。其作为扩大优质种畜资源的重要手段,现在已在全世界范围得到广泛应用,并从鲜胚移植发展到冻胚移植,从整胎移植到分割胚、     

纯种和牛快速扩繁关键技术研究

为达到快速扩繁纯种和牛的目的,试验从供体牛高强度超数排卵、胚胎性别鉴定及不同品种受体牛移植对产犊、妊娠期和犊牛初生重的影响进行研究。结果表明,纯种和牛连续超排9次,每次间隔30 d,第2次头均回收胚数22.78枚,显著高于第5~9次结果（P< 0.05）,其余各组之间无显著差异（P> 0.05）;第1~3次头均可用胚数分别为8.67、13.78、8.56枚,组间无显著差异（P> 0.05）,第2次头均可用胚数显著高于第4~9次头均可用胚数（P< 0.05）,极显著高于第7、9次头均可用胚数（P< 0.01）。性别鉴定胚胎和常规胚胎移植妊娠率和产犊率差异均不显著（P> 0.05）,性别鉴定胚胎的母犊率为95.56%。西门塔尔杂交牛受体产犊率高于和牛杂交牛、荷斯坦奶牛受体,但差异不显著（P> 0.05）。和牛杂交牛受体妊娠期极显著高于西门塔尔杂交牛和荷斯坦奶牛受体（P< 0.01）,西门塔尔杂交牛和荷斯坦奶牛之间差异不显著（P> 0.05）;西门塔尔杂交牛受体所产和牛母犊初生重显著高于和牛杂交牛受体（P< 0.05）,极显著高于荷斯坦奶牛受体（P< 0.01）,3个品种受体牛产和牛公犊初生重之间差异不显著（P> 0.05）;同一品种受体牛其产公、母犊牛的妊娠期及初生重均相近。     

我省第一头胚胎分割移植奶牛犊诞生

黑龙江省畜牧研究所家畜繁殖研究室全体科研人员在牛冷冻胚胎移植妊娠率达39%的基础上,又攻克了奶牛胚胎分割移植技术难关。今年4月3日,农户刘永贵饲养的杂种牛顺利分娩,生下一头黑白花母犊,健康活泼,发育良好(见照片)。这是我省首例分割胚胎产下的犊牛。其余怀孕的受体牛将在近期内分娩。照片说明:图为齐齐哈尔市郊哈力乡后平村农民刘永贵饲养的一头杂种母牛。年龄6岁,黑毛色。1988年7月9日移入1对黑白花奶牛分割胚胎,经270天妊娠,产下了1头健壮活泼的黑白花母犊牛。     

移植奶牛胚胎冷冻解冻后分割成的 二分胚生产同卵双生犊牛的实验

对解冻后品质优良的A级囊胚进行分割,并将二分胚移植生产同卵双生犊牛。结果表明,9枚A级囊胚切割成18枚半胚,胚胎分割成功率100%（9/9）。单胚移植受体6头,妊娠2头,妊娠率33.3%;双半胚移植6头,妊娠2头,妊娠率33.3%。     

我国奶牛胚胎SRY性鉴试验获新成果

继我国科学工作者1991年在国际上首次采用扩增牛的 SRY 基因鉴定牛胚胎性别获得成功之后,1992年北京农学院吴学清和高建明等研究人员在江苏连云港东辛农场奶牛公司牛场进行胚胎性别 SRY 鉴别技术的扩大试验中,在当地生产条件下,选用741号牛(4岁、2胎次)超数排卵,一次采集胚胎8个,镜检可用胚7个,不可用胚1个。将切割少量取样的待检鲜胚,于当日分别移植给7头受体母牛,两个月时妊检确认6头妊娠。孕牛中2头因故流产和因病淘汰外,其余4头于1993年1月份相继产犊。其中公犊3头,母犊1头,与胚样性别检测结果相符合。该供体于超排后40天再次妊娠,1993     

An Inter‐Subspecies Cloned Buffalo (Bubalus bubalis) Obtained by Transferring of Cryopreserved Embryos via Somatic Cell Nuclear Transfer

The aim of this study was to explore the feasibility of cryopreservation of inter‐subspecies cloned embryos in buffalo. In our experiment, river buffalo ear fibroblast nucleus was fused into swamp buffalo oocyte cytoplasm. The blastocyst formation rate for nuclear transfer of freshly thawed cells was not different from those of growing cells, confluent or serum‐starved cells. A total of 122 cloned blastocysts derived from cryopreserved fibroblasts were cryopreserved and thawed, 37 were survived, the cryosurvival rate was 30.3%. The survived blastocysts were transferred into 15 recipient buffalos. Five of the recipients established pregnancy, but four of them aborted on day 53, 59, 145 and 179 of gestation respectively. One cross‐bred buffalo (Murrah × Swamp buffalo (2n = 49) received three embryos delivered a 40.5 kg female calf by natural delivery on day 320 of gestation. Up to now (13‐month old), the cloned calf has been growing well with no abnormity observed. These results demonstrated that cryopreservation of inter‐subspecies cloned embryos is feasible to produce buffalo offspring.     

Migration and differentiation of gonadal germ cells under cross-sex germline chimeras condition in domestic chickens

A series of experiments was conducted to investigate migration, proliferation and differentiation of gonadal germ cells (GGCs) collected from the gonads of 7-day-old chick embryos under cross-sex germline chimera conditions. The migratory and proliferative abilities of exogenous GGCs were examined by transferring 50 fluorescently labeled GGCs collected from White Leghorn (WL) embryos into the blood of 2-day-old Rhode Island Red (RIR) embryos. No significant difference was observed in the number of fluorescently labeled GGCs in the gonads of recipient embryos among any of the four possible donor and recipient sex combinations. Cross-sex germline chimeras were produced to examine the differentiation of GGCs by transferring 100 GGCs from WL embryos into 2-day-old RIR embryos. Exogenous-GGC-derived progeny were obtained from both male and female recipients, except when female GGCs were transferred into male recipients. The migratory ability of GGCs recovered from the 7-day-old embryonic gonad was not influenced by cross-sex germ cell transfer conditions, whereas the differentiation of the GGCs was affected by the sex combinations of GGCs donors and recipients.     

Successful Production of Piglets Derived from Expanded Blastocysts Vitrified Using a Micro Volume Air Cooling Method without Direct Exposure to Liquid Nitrogen

This study was conducted to clarify the feasibility of newly developed vitrification techniques for porcine embryos using the micro volume air cooling (MVAC) method without direct contact with liquid nitrogen (LN₂). Expanded blastocysts were vitrified in a solution containing 6 M ethylene glycol, 0.6 M trehalose and 2% (wt/vol) polyethylene glycol in 10% HEPES-buffered PZM-5. The blastocysts were collected from gilts and vitrified using the new device (MVAC) or a Cryotop (CT). Blastocysts were stored in LN₂ for at least 1 month. After warming, cryoprotective agents were removed using a single step. Survival of the embryos was assessed by in vitro culture (Experiment 1) and by embryo transfer to recipients (Experiment 2). In Experiment 1, the embryos vitrified by the MVAC or CT and fresh embryos without vitrification (Control) were used. The survival rates of embryos in the MVAC, CT and Control groups were 88.9% (32/36), 91.7% (33/36) and 100% (34/34), respectively, after 48 h culture, and the hatching rates of embryos after 48 h incubation were 69.4% (25/36), 63.9% (23/36) and 94.1% (32/34), respectively. In Experiment 2, 64 vitrified embryos were transferred to 5 recipient gilts, and 8 healthy piglets were produced from 3 recipients in the MVAC group. Similarly, 66 vitrified embryos were transferred to 5 recipient gilts, and 9 healthy piglets were produced from 2 recipients in the CT group. These results indicated that porcine expanded blastocysts can be cryopreserved using the MVAC method without potential pathogen contamination from LN₂.     

家兔胚胎细胞核移植与连续移植

将发育不同时期的兔胚和移核胚的卵裂球细胞核与去核的成熟卵母细胞共同组成移核胚,通过中间受体培养和移植实验检验胚胎的发育能力。结果表明,（１）兔囊胚之前各个时期的胚胎细胞核均可使移核胚发育到囊胚;（２）胚胎极化前后的卵裂球参与组成的移核胚发育到囊胚的比例无显著差异。但极化后 ６４细胞胚的卵裂球与去核卵母细胞的融合率低于极化前的 ８细胞胚胎卵裂球;（３）兔 １６细胞胚与去核的卵母细胞组成的移核胚可以发育到期,产仔率为 ３．１６％ ;（４）兔移核胚卵裂球用于连续核移植,其后 ２ 代均可发育成囊胚,其中第 １ 代移核胚与第 ２ 代移核胚发育率相似,但显著高于第 ３ 代移核胚;（５）兔移核胚和各代连续移核胚卵裂球与去核卵的融合率无显著差异。     

胚胎质量和发育阶段对奶牛胚胎移植妊娠率的影响

[目的]为了评估胚胎质量和发育阶段对奶牛胚胎移植妊娠率的影响。[方法]使用63头青年奶牛作为供体进行超数排卵,评估回收胚胎质量和发育阶段。选择334头青年奶牛作为受体鲜胚移植不同质量和发育阶段胚胎。对胚胎质量分布、发育阶段分布、不同质量胚胎和不同发育阶段胚胎移植30 d妊娠率进行统计分析。[结果]可用胚胎中A级胚胎比例(60.78%)显著高于B级和C级胚胎比例(36.70%和2.52%)(P<0.05);致密桑椹胚比例(54.36%)显著高于早期囊胚,囊胚和扩张囊胚比例(18.35%,25.0%和2.29%)(P<0.05)。A级和B级胚胎移植30 d妊娠率(63.55%和64.35%)显著高于C级胚胎移植30 d妊娠率(44.44%)(P<0.05);致密桑椹胚、早期囊胚、囊胚和扩张囊胚移植30 d妊娠率差异不显著(P<0.05),早期囊胚、囊胚移植30 d妊娠率高于致密桑椹胚、扩张囊胚移植30 d妊娠率(P<0.05)。[结论]选择不同发育阶段的A级和B级胚胎能够获得较高胚胎移植妊娠率,增加早期囊胚和囊胚阶段胚胎移植数量能够提高胚胎移植妊娠率。     

Use of embryo transfer to induce twinning in beef cattle: embryo survival rate, gestation length, birth weight and weaning weight of calves

Experiments were conducted in 1985 and 1986 at the Eastern Ohio Resource Development Center, Belle Valley, to examine the feasibility of using embryo transfer to induce twinning and to examine the influence of twinning on traits of the cow and calf. Embryos were collected from a total of 14 superovulated Angus donors on two dates each in 1985 and 1986 and were transferred to Angus recipients. A total of 124 embryos were transferred to 79 recipients, with 43 (34.7%) calves born alive. Seven of 45 (15.6%) recipients implanted with two embryos produced twins. In no case did both halves of the 15 embryos that were split to produce identical twins and implanted in the same recipient survive to birth. Proportion of calves born alive did not differ among transfer codes 3 (nonsplit embryos from two different donors implanted in separate uterine horns of the same recipient), 6 (nonsplit embryos from one embryo flush implanted in separate uterine horns of the same recipient) and 7 (nonsplit embryos from two different donors implanted in the same uterine horn of one recipient). Surgical transfers tended to result in a higher proportion of embryos surviving to birth (.43 vs .21; P = .16) and a higher twinning rate (.29 vs .04; P = .36) than did nonsurgical transfers. Age of recipient did not influence embryo survival (P = .98) or twinning rate (P = .99). Gestation length was 5 d shorter (P less than .01) for twin calves than for singles. Singles were 9 kg heavier (P less than .01) at birth and 32 kg heavier (P less than .01) at weaning than twins. However, cows raising twins produced 108 kg (51%) more total weaning weight than did cows raising singles.     

Multiple embryo-transplant offspring produced from quartering a bovine embryo at the morula stage

A non-surgical embryo collection was completed on a day 7 superovulated Chianina donor cow. Because all but two of the ova from the collection were unfertilised and a surplus of potential recipients was available, one embryo (an excellent quality late morula) was dissected into four equal portioned 'quarter' embryos using a simplified micromanipulation procedure. Each quarter embryo was then placed in a 0.25 ml French straw and non-surgically transplanted to four different crossbred beef recipient females. The remaining embryo was similarly transplanted to a herd mate recipient as an intact embryo. One recipient returned to oestrus, one recipient had an extended post transfer cycle and the two remaining recipients produced a live quarter embryo transplant calf each within 24 hours of the other. The intact embryo placed in a herd mate recipient did not produce a transplant calf. To the authors' knowledge, these transplant offspring are the first live births reported from a non-surgically collected later-stage bovine morula (day 7), which had been dissected into quarters and then individually transplanted non-surgically to recipient females. The procedure was relatively simple to perform and was completed in less than one hour.     

Production of HanWoo (Bos taurus coreanae) fetuses following interbreed somatic cell nuclear transfer

The possibility of producing HanWoo (Bos taurus coreanae) calves from transferable bovine embryos, obtained by interbreed nuclear transfer using Holstein cytoplasts and surrogates, was investigated. As donor nuclei, HanWoo fetal fibroblasts were used. Cells were induced into quiescence by serum deprivation for 4-7 days before nuclear transfer. In vitro matured Holstein oocytes were enucleated, and single donor cell was placed into the perivitelline space of enucleated oocyte. After reconstruction, the embryos were fused. activated and cultured. On day 7, the embryos that developed to the blastocyst stages were transferred into Holstein recipient cows on day 6 to 7 of estrous cycle (estrus=0). The reconstructed embryos were successfully fused (58.8%; 47/80), cleaved (91.5%; 43/47), and developed to blastocysts (29.8%; 14/47). Eleven blastocysts were transferred into 5 Holstein recipient cows. Two recipients were pregnant, confirmed by ultrasonography at day 60 of gestation. But, one of them was opened between on day 80 to 100 of pregnancy, and the other had a stillbirth on day 255. The stillborn calf was physically normal, and we couldn't find any evidence of anomaly. These results show that cells derived from HanWoo somatic cell lines can be reprogrammed by interbreed nuclear transfer and develop subsequently in vivo as well as in vitro.     

Effects of recloning on the efficiency of production of alpha 1,3-galactosyltransferase knockout pigs

Obtaining sufficient transgenic cells via selective cultivation of genetically manipulated somatic cells is difficult due to the limited number of cell divisions. Additionally, if irreversible mutations in a cell's chromosomes occur during selective cultivation and the cell is used as the nuclear donor, somatic cell nuclear transfer (SCNT) embryos often exhibit abnormal development. On the other hand, a SCNT method in which fetal cells derived from SCNT embryos are used as the nuclear donor (recloning method) is an effective technique for obtaining large quantities of transgenic cells. In this study, we compared the in vivo development rate of SCNT embryos produced from porcine alpha1-3 galactosyltransferase gene knockout (GTKO) cells by a recloning method with that of SCNT embryos produced without recloning from porcine GTKO cells (direct method). In the direct method, 557 and 462 cloned embryos were produced using two types of activation methods, the two-step activation (TA) method and the delayed activation (DA) method, and then transferred into 6 and 4 recipients, respectively, but no piglets were born from these recipients. In the recloning method, 956 and 1038 cloned embryos were produced using the TA and DA methods, respectively, and then transferred to 8 and 7 recipients, respectively. Two piglets were born from one recipient in the TA group and 6 piglets were born from 3 recipients in the DA group. This report indicates that the recloning method improved the developmental capacity of SCNT embryos reconstructed with gene-targeted somatic cells.     

波尔山羊胚胎数量、质量与受体移植妊娠率的关系

本文就波尔山羊胚胎数量，质量与受体移植妊娠率的关系进行了探讨。结果表明：胚胎质量优劣是直接影响胚胎移植成功率的关键因素。移植胚胎数量因品种不同有差异。移植双胚，奶山羊受体胚胎成羔率53．4％，明显高于成都麻羊胚胎成羔率36．3％，成都麻羊单胚成羔率为66．7％。