Production of cloned goats after nuclear transfer using adult somatic cells.

The developmental potential of adult somatic nuclei after nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated in a dwarf breed of goat (BELE: Breed Early Lactate Early). Somatic donor cells were obtained from two different sources: 1) adult granulosa cells (GCs) and 2) fetal fibroblasts. Primary GCs were obtained from follicular aspirants after laparoscopic oocyte pick-up (LOPU) and were cryopreserved immediately. Frozen aliquots of cells were thawed and cultured until confluent and were then cultured in low serum for 4 days before use in NT. Immature oocytes were obtained by LOPU and matured before enucleation and NT. Ninety-one adult GC-derived NT embryos were transferred into eight recipients, four of which were confirmed pregnant (50%) at Day 30 by ultrasound. Fifty-four male fetal fibroblast-derived NT embryos were transferred into six recipients, one of which was confirmed pregnant (17%). All pregnancies were maintained through term. Four recipients delivered seven female kids (three sets of twins) derived from the GC cultures (7.7% of embryos transferred). The other recipient delivered two male kids (3.7% of embryos transferred). Birth weights were within the normal range for dwarf goats. One female twin and one male twin died at birth; the remaining kids appeared healthy and normal. DNA analysis confirmed that the kids were genetically identical to their respective donors. These results demonstrated that adult caprine somatic cells could direct normal development after NT.     

山羊品种间体细胞核移植研究

萨能奶山羊是著名的奶用山羊品种,波尔山羊则是世界著名的肉用山羊品种。为了研究波尔山羊体细胞在奶山羊卵母细胞中的去分化,我们针成年波尔山羊的颗粒细胞或耳皮肤成纤维细胞作为供核细胞（试验组）,移入奶山羊中Ⅱ期的去核卵母细胞透明带下,经电融合和离子霉素与6－二甲基氨基嘌呤－DMAP）激活,直接移入同期发情奶山羊输卵管或经体内培养,将发育的重构胚移入同期发情羊子宫内。妊娠早期作B超诊断,确立妊娠的观察至足月。同时将奶山羊的35日龄胎儿成纤维细胞作供核细胞（对照组）,按试验组同样方法处理,将重构胚直接移入同期发情的奶山羊输卵管内。结果：试验组,波尔羊颗粒粒细胞与耳皮肤成纤维2细胞的融合率分别为78．2％（115／147）,57．4％（116／202）,重构胚卵裂率为85．8％（115／134）,桑椹胚,囊胚的发育率38．8％（52／134）,早期妊娠三头,分别于妊娠40,60,60日龄终止妊娠。对照组,融合率为89．5％（136／152）,早期妊娠率为42．9％（6／14）,四头受体足月分娩,产四头公羊羔,其中三头存活,一头分娩时死于肺不扩张,并体重过大,显示胎儿过大综合症。经基因型鉴定证实,这四头克隆羔羊均源于同一胎儿成纤维细胞系。以上结果表明,波尔羊体细胞核在奶山羊卵母细胞中能够去分化,并维持一定程度的发育。     

Viable Transgenic Goats Derived from Skin Cells

The current study was undertaken to evaluate the possibility of expanding transgenic goat herds by means of somatic cell nuclear transfer (NT) using transgenic goat cells as nucleus donors. Skin cells from adult, transgenic goats were first synchronized at quiescent stage (G0) by serum starvation and then induced to exit G0 and proceed into G1. Oocytes collected from superovulated donors were enucleated, karyoplast-cytoplast couplets were constructed, and then fused and activated simultaneously by a single electrical pulse. Fused couplets were either co-cultured with oviductal cells in TCM-199 medium (in vitro culture) or transferred to intermediate recipient goat oviducts (in vivo culture) until final transfer. The resulting morulae and blastocysts were transferred to the final recipients. Pregnancies were confirmed by ultrasonography 25-30 days after embryo transfer. In vitro cultured NT embryos developed to morulae and blastocyst stages but did not produce any pregnancies while 30% (6/20) of the in vivo derived morulae and blastocysts produced pregnancies. Two of these pregnancies were resorbed early in gestation. Of the four recipients that maintained pregnancies to term, two delivered dead fetuses 2-3 days after their due dates, and two recipients gave birth to healthy kids at term. Fluorescence in situ hybridization (FISH) analysis confirmed that both kids were transgenic and had integration sites consistent with those observed in the adult cell line.     

山羊品种间体细胞核移植研究

萨能奶山羊是著名的奶用山羊品种,波尔山羊则是世界著名的肉用山羊品种.为了研究波尔山羊体细胞在奶山羊卵母细胞中的去分化,我们将成年波尔山羊的颗粒细胞或耳皮肤成纤维细胞作为供核细胞(试验组),移入奶山羊中Ⅱ期的去核卵母细胞透明带下,经电融合和离子霉素与6-二甲基氨基嘌呤(6-DMAP)激活,直接移入同期发情奶山羊输卵管或经体内培养,将发育的重构胚移人同期发情羊子宫内.妊娠早期作B超诊断,确立妊娠的观察至足月.同时将奶山羊的35日龄胎儿成纤维细胞作供核细胞(对照组),按试验组同样方法处理,将重构胚直接移入同期发情的奶山羊输卵管内.结果试验组,波尔羊颗粒粒细胞与耳皮肤成纤维细胞的融合率分别为78.2%(115/147)、57.4%(116/202),重构胚卵裂率为85.8%(115/134),桑椹胚、囊胚的发育率38.8%(52/134),早期妊娠三头,分别于妊娠40、60、60日龄终止妊娠.对照组,融合率为89.5%(136/152),早期妊娠率为42.9%(6/14),四头受体足月分娩,产四头公羊羔,其中三头存活,一头分娩时死于肺不扩张,并体重过大,显示胎儿过大综合症.经基因型鉴定证实,这四头克隆羔羊均源于同一胎儿成纤维细胞系.以上结果表明,波尔羊体细胞核在奶山羊卵母细胞中能够去分化,并维持一定程度的发育.     

Effect of cytoplast on the development of inter-subspecies nuclear transfer reconstructed goat embryo

The aim of this study was to investigate effect of cytoplast on the development competence of reconstructed embryos derived from inter-subspecies somatic cell nucleus transfer (SCNT). First, the development potency of reconstructed embryos produced by transferring Boer goat fibroblast cell nucleus of different ages into enucleated Sannen goat ova was evaluated in order to determine which age of nuclear donor is favorable for the reconstructed embryos development. Secondly, the another component of reconstructed embryos, "cytoplast," was evaluated by comparing the effect of ovum cytoplast derived from Sannen male symbol x Boer female symbol descendant on the reconstructed embryos development to that of Sannen goat ovum cytoplast. The results revealed that the development rate of the reconstructed embryos derived from 2 months old Boer goat somatic cells was the highest, their gestation rate was up to 50%, and one viable male offspring was obtained. The cytoplast derived from the crossbreeding goats improves the development competence of reconstructed embryos, which birth rate was 5.5%. The genetic identification of offspring by using PCR-SSCP analysis confirmed that these cloned kids were derived from the donor. The results above reveal that the cytoplast of Sannen goat ovum could induce the dedifferentiation of somatic cell nuclei derived from Boer goat, but the reprogramming process of these reconstructed embryos seems incomplete, probably due to some incorrect processes happened after implantation. Relatedness components of nucleus donor in cytoplast of the crossbreeding goat may be helpful to induce the dedifferentiation of somatic cell nuclei completely and improve the development competence of the reconstructed embryos.     

Improvement of the nuclear transfer efficiency by using the same genetic background of recipient oocytes as the somatic donor cells in goats

We have compared the effect of the genetic background of recipient oocytes on the in vitro and in vivo development of nuclear transfer reconstructed embryos in goats. Adult fibroblast cells from Boer goats were used as donor cells, and recipient oocytes were obtained from Boer goats and Boer cross-breeds (Boer♂×Huanghuai♀). Nuclear transfer reconstructed embryos were cultured in vitro, or transferred into recipient goats. The mitochondrial origin of 2 cloned Boer goats was investigated by analysing the D-loop region based on polymorphisms via DNA sequencing. There was no significant difference in the fusion rate and cleavage rate of reconstructed embryos (P>0.05), when using Boer and cross-breeding goat oocytes as recipient cytoplast respectively. However, in vitro morula development of reconstructed embryos from Boer oocytes was significantly higher than that of cross-breeding embryos (34.1% versus 19.1%, P<0.05). There was no significant difference in the rate of pregnancy and foetus loss between the 2 breeds. However, the live-birth rate was significantly higher with Boer goat oocyte recipients than the cross-breeds (3.1% versus 0.8%, P<0.05). Mitochondrial analysis showed that the 2 cloned goats were similar to their respective oocyte donor goats, and significantly different from the nucleus donor. In conclusion, genetic background of recipient oocytes affected in vitro and in vivo development of reconstructed embryos, with the homologous background of cytoplast and nuclear donor benefiting development of reconstructed embryos. The mitochondrial origin of the 2 cloned Boer goats came from recipient oocytes, not donors.     

以经过转染的乳腺上皮细胞生产克隆羊

为研究转基因乳腺上皮细胞发育的全能性,利用电转染方法将人乳铁蛋白(hLF)乳腺特异性表达载体电转染山羊乳腺上皮细胞,经G418和PCR筛选获得阳性克隆细胞株,经催乳素诱导的细胞株上清液用Western blotting方法检测hLF的表达。以转基因与上清液中表达hLF均为阳性的细胞为核供体细胞,进行山羊体细胞核移植。结果为:16株细胞表达重组hLF,分子质量为75 kD;将144枚重构胚移入16只同步发情的山羊输卵管中,在移植后的30 d、60 d和90 d的妊娠率分别为87.5%、81.3%和62.5%;最终3只受体妊娠足月,产下3只克隆羊,克隆效率为2.1%,PCR-RFLP分析表明克隆羊均来自供体羊细胞,但没有整合外源基因。结果表明,hLF转基因乳腺上皮细胞能分泌hLF;乳腺上皮细胞经转染、筛选和长期培养的条件下,能保持发育的全能性。     

Selection of in vitro produced, transgenic embryos by nested PCR for efficient production of transgenic goats

The production of valuable pharmaceutical proteins using transgenic animals as bioreactors has become one of the goals of biotechnology. However, the efficiency of producing transgenic animals by means of pronuclear microinjection is low. This may be attributed in part to the low integration rate of foreign DNA. Therefore, a large number of recipients are required to produce transgenic animals. We recently developed a transgenic procedure that combined the techniques of goat oocyte in vitro maturation (IVM), in vitro fertilization (IVF), microinjection, preimplantation selection of the transgenic embryos with nested PCR and transferring the transgenic embryos into the recipient goat uterus to produce transgenic goats. Thirty-seven transgenic embryos determined by nested PCR were transferred to thirty-two recipient goats. In the end, four live-born kids were produced. As predicted, all the live kids were transgenic as identified by PCR as well as Southern blot hybridization, The integration rate was 100% (4/4) which was completely in accordance with the results of embryo preimplantation detection. The results showed a significant decrease in the number of recipients required as only 8 recipients (32/4) were needed to obtain one live transgenic goat. We suggest that the transgenic system described herein may provide an improved way to efficiently produce transgenic goats on a large scale.     

Birth of normal kids after microinjection of pronuclear embryos in a transgenic goat (Capra hircus) production program in Brazil

This pilot project was designed to determine if normal kids could be produced after microinjection in pronuclear embryos and subsequent transfer to recipients in a transgenic goat program in Brazil. Twelve donors of the Saanen breed and 17 recipients of an undefined breed were used. The estrus of both donors and recipients was synchronized by a standard progestagen treatment and superovulation obtained by six pFSH injections. Donors in estrus were mated with fertile Saanen bucks. Zygotes were recovered surgically by flushing oviducts. The recovered zygotes with visible pronuclei were microinjected with 500 to 1000 copies of the human G-CSF gene. Two or four embryos were surgically transferred into the oviducts of recipients. One recipient became pregnant and two kids were born. No transgenic goat was identified after PCR analysis. Even though transgenic goats were not obtained, this experiment establishes the basis of a synchronization and superovulation regimen for use in goats raised in Brazil, for the purpose of collecting and manipulating the pronuclear embryos. This project also showed that microinjected one-cell goat embryos can survive to produce live young following surgical transfer.     

Generation of dwarf goat (Capra hircus) clones following nuclear transfer with transfected and nontransfected fetal fibroblasts and in vitro-matured oocytes

The developmental potential of caprine fetal fibroblast nuclei after in vitro transfection and nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated. Fetal fibroblasts were isolated from Day 27 to Day 30 fetuses from a dwarf breed of goat (BELE: breed early lactate early). Cells were transfected with constructs containing the enhanced green fluorescent protein (eGFP) and neomycin resistance genes and were selected with G418. Three eGFP lines and one nontransfected line were used as donor cells in NT. Donor cells were cultured in Dulbecco minimum Eagle medium plus 0.5% fetal calf serum for 4-8 days prior to use in NT. Immature oocytes were recovered by laparoscopic ovum pick-up and matured for 24 h prior to enucleation and NT. Reconstructed embryos were transferred as cleaved embryos into synchronized recipients. A total of 27 embryos derived from transgenic cells and 70 embryos derived from nontransgenic cells were transferred into 13 recipients. Five recipients (38%) were confirmed pregnant at Day 35 by ultrasound. Of these, four recipients delivered five male kids (7.1% of embryos transferred) derived from the nontransfected line. One recipient delivered a female kid derived from an eGFP line (7.7% of embryos transferred for that cell line). Presence of the eGFP transgene was confirmed by polymerase chain reaction, Southern blotting, and fluorescent in situ hybridization analyses. Nuclear transfer derivation from the donor cells was confirmed by single-strand confirmation polymorphism analysis. These results demonstrate that both in vitro-transfected and nontransfected caprine fetal fibroblasts can direct full-term development following NT.     

Efficiency of donor cell preparation and recipient oocyte source for production of transgenic cloned dairy goats harboring human lactoferrin

The objective was to investigate the effects of the transgenic donor cell synchronization method, oocyte sources, and other factors, on production of hLF-gene nucleus transfer dairy goats. Three transfected cell lines from ear biopsies from three 3-mo-old Saanen dairy goats (designated Number 1, Number 2, and Number 3, respectively) were selected as karyoplast donors for somatic cell nuclear transfer (SCNT) after detailed identification (including PCR and sequencing of PCR products). In donor cell cycle synchronization studies, the apoptosis rate of hLF transgenic fibroblasts was not different (P > 0.05) after 3 days of serum starvation or 2 days of contact inhibition. Additionally, there was no effect (P > 0.05) on developmental capacity of reconstructed embryos; however, the kidding rate of recipients in the serum starvation group was higher than that in the contact inhibition group (18 vs. 0%, respectively). The production efficiency of the transgenic cloned goats using donor cells from the Number 1 dairy goat cell line was higher than those using the Number 2 and the Number 3 cell lines (kidding rates were 18, 2, and 0%, respectively, P < 0.05). The oocyte source did not significantly affect the pregnancy rate of hLF-transgenic cloned dairy goats, but more fetuses were aborted when using in vitro matured oocytes compared to in vivo matured oocytes. In summary, utilizing transfected 3-mo-old dairy goat fibroblasts as donor cells, seven live offspring were produced, and the hLF gene was successfully integrated. This study provided additional insights into preparation of donor cells and recipient oocytes for producing transgenic cloned goats through SCNT.     

Production of transgenic goats by pronuclear microinjection of in vitro produced zygotes derived from oocytes recovered by laparoscopy

Oocytes collected by laparoscopic ovum pick-up (LOPU) were successfully used to produce transgenic goats by pronuclear microinjection of in vitro zygotes. Estrus cycles of 109 donor goats were synchronized using intravaginal sponges impregnated with 60 mg of medroxyprogesterone acetate and treatment with 70 mg NIH-FSH-P1 and 300 IU eCG to stimulate follicular development. Follicles were aspirated under laparoscopic observation. In vitro maturation (IVM) of oocytes was performed in M199 supplemented with hormones, kanamycin and 10% estrus goat serum. Following IVM, oocytes were cocultured with capacitated semen in TALP supplemented with 20% estrus goat serum for 15-20 h. The resulting zygotes were microinjected with a linear DNA fragment. In total, 3293 follicles were aspirated (15.7+/-9 follicles aspirated per donor) and 2823 oocytes were recovered (13.4+/-8 oocytes per donor). A total of 1366 zygotes were microinjected and transferred into 219 recipient goats by midventral laparotomy (average 6.2 embryos per recipient). A total of 150 kids were born, of which 9 (6 M: 3 F) were confirmed to be transgenic by PCR and Southern blotting analyses. These results demonstrate that acceptable transgenesis rates can be obtained in goats by DNA microinjection of in vitro produced zygotes.     

继代核移植能成倍提高来自奶山羊卵胞质体的异种间核移植重构胚的发育率

为了提高异种间核移植重构胚的发育率,本研究以体内排放的奶山羊成熟卵为供胞质的受体细胞,以人、兔、波尔山羊等的异种或亚种体细胞的原代核移植（Primary Somatic Cell Nuclear Transfer,PSCNT）重构早胚（8-16细胞期）的卵裂球作供核体,观察经亚种或异种卵胞质体短期“修饰”的核再移植产生的继代（Secondary SCNT,SSCNT）重构胚的着床前发育潜能。结果：人、兔、波尔山羊的继代桑椹／囊胚发育率均显著地高于其PSCNT胚胎（人,14．81％VS．7．79％;兔,23．53％VS．12．50％;波尔羊,55．35％VS．24．53％）;这些早胚的各阶段发育时程仍遵循供核体动物正常受精卵的发育时程。结果启示：奶山羊成熟卵胞质对异种体细胞核亦具一定的去分化能力,能支持重构胚发育到囊胚;异种重构胚的发育特征是由供体核所决定的;继代核移植几乎能够成倍提高异种间重构胚的着床前发育率,提示核的去分化完全是在母型信息主导的调控之下完成的,而进一步发育的时序似乎是由核决定的：成倍延长在含母型信息主导调控环境中的时间能成倍提高SCNT重构胚的着床前发育率。     

由成年转基因山羊体细胞而来的克隆山羊

在已经获得的乳腺特异性表达人促红细胞生成素 (rhEPO)成年转基因山羊 (Caprahircus)的基础上 ,取其耳尖成纤维细胞和卵巢颗粒细胞 ,进行体外传代培养 ,然后将这种培养的转基因山羊的体细胞移入去核的处于第Ⅱ次减数分裂中期的卵母细胞中 ,并进行电融合 ,构建重构胚胎 ,重构胚胎在体内培养 6d ,再将发育至囊胚或桑椹胚的重构胚胎移入同步情期的寄母羊子宫内。结果 ,有 2只寄母羊妊娠并最终产下 2只成活的克隆山羊。她们分别来自同一成年母羊的耳尖成纤维细胞和卵巢颗粒细胞。克隆羊经PCR RFLP图谱分析显示 :以克隆羊组织DNA为模板的PCR产物与相应的提供体细胞的基因羊的PCR产物的酶切图谱完全一致 ;并且经PCR对外源hEPO基因检测表明 2只克隆山羊均携带hEPO外源基因。由此证明获得了转基因成年体细胞的克隆山羊     

Production, freezing and transfer of embryos from a bluetongue-infected goat herd without bluetongue transmission

In order to import non-seasonal Creole goats from the Carribean to Europe for an experimental purpose, thirty Creole goats were treated with 10 mg of FSH; embryos were collected at slaughter, washed and deep frozen. After rapid thawing, they were reimplanted surgically into European dairy goats. Twenty-four females ovulated but only 17 of the ovulating females had functional corpora lutea (CL) at collection. Ovulation rate (CL goat ) and recovery rate (embryo CL ) were 13.8 and 78% for females with functional CL. Of 191 embryonic structures collected, 79% were considered suitable for deep freezing: 23% were young blastocysts, 47% were expanded blastocysts, and 30% were zona-pellucida (zp)-free and zp-damaged embryos. Seventy-eight embryos were thawed and 63 were reimplanted. Sixty-eight percent of the recipient females delivered 19 kids. The percentage of kids born relative to good-quality re-implanted embryos was higher for zp-free embryos (64%) than for young and expanded blastocyts (36%). Forty-seven percent of the donor females had strong positive serological reactions for bluetongue virus antibodies against serotypes 6 and 14. However, no recipient goats or newborn kids were positive. Virus isolation attempts on the collection media and last embryo washes were negative.     

单、双标记基因筛选的转人乳铁蛋白基因供核细胞生产克隆山羊效率的比较

为比较两种筛选标记基因生产转人乳铁蛋白(hLF)基因克隆山羊的效率,利用单(新霉素抗性基因,Neor)、双(新霉素抗性和绿色荧光蛋白基因,Neor/GFP)标记基因筛选转基因的供核细胞,并制作体细胞核移植转基因山羊。山羊胎儿成纤维细胞电转染单标记基因表达载体(pBLC14)或双标记基因表达载体(pAPLM),分别有58.8%(20/34)和86.7%(26/30)的抗性细胞株检测到外源基因;转染pAPLM的细胞传代培养后,仅有20%(6/30)株细胞在传代中所有细胞均能观察到荧光;分别以pBLC14和pAPLM的细胞株作为供核细胞进行体细胞核移植,共获得806枚重构胚胎,胚胎移植受体后35 d、60 d妊娠率分别为53.8%、26.9%和39.1%、21.7%,最终分别产下5只(1.9%)和7只(1.4%)克隆山羊;经PCR及Southern blotting检测,所有出生山羊均整合有外源基因。结果显示,以单、双标记基因筛选供核细胞,其重构胚融合率、怀孕率和克隆动物出生率差异不显著(P>0.05),Neor/GFP双标记基因能准确、有效地用于转基因供核细胞筛选。同时,结果也表明Neor/GFP双标记基因转染的体细胞作为供核细胞对体细胞克隆效率未出现不利影响。     

Cloned goats (Capra hircus) from adult ear cells

The average number of available oocytes recovered per ovary collected during the breeding season in dairy goats was 5.5 (1815/330). 66.17% (1201/1815) of oocytes extruded the first polar body after maturation in vitro for 20 h. 75.44% (906/1201) of matured oocytes with membrane evagination around the MII chromosomes were enucleated. Ear skin fibroblast cells were derived from an adult female dining Grey goat (C. hircus). The cells were cryopreserved in liquid nitrogen after passage 2. Thawed cells were further cultured for 3-6 passages and were subjected to serum starvation by 0.5% FBS for 2-10 d, then used as donor cells for nuclear transfer. 98.12% (889/906) of the enucleated oocytes were reconstructed by intracytoplasmic injection of karyoplast. The reconstructed embryos were activated by 5μ mol/L ionomycin for 4.5 min and further activated by culturing with 6-dimethylaminopurine (6-DMAP) for 3 h. After 36 h of culture in mCR1aaBF, 76.69% (645/841) of the cloned embryos cleaved. There were no signifi     

Cloned goats (Capra hircus) from adult ear cells

The average number of available oocytes recovered per ovary collected during the breeding season in dairy goats was 5.5 (1815/330). 66.17% (1201/1815) of oocytes extruded the first polar body after maturation in vitro for 20 h. 75.44% (906/1201) of matured oocytes with membrane evagination around the MⅡchromosomes were enucleated. Ear skin fibroblast cells were derived from an adult female Jining Grey goat (C. hircus). The cells were cryopreserved in liquid nitrogen after passage 2. Thawed cells were further cultured for 3-6 passages and were subjected to serum starvation by 0.5% FBS for 2-10 d, then used as donor cells for nuclear transfer. 98.12% (889/906) of the enucleated oocytes were reconstructed by intracytoplasmic injection of karyoplast. The reconstructed embryos were activated by 5 μmol/L ionomycin for 4.5 min and further activated by culturing with 6-dimethylaminopurine (6-DMAP) for 3 h. After 36 h of culture in mCR1aaBF, 76.69% (645/841) of the cloned embryos cleaved. There were no significant differences in development in vitro between the cloned embryos derived from donor cells precooled at 4℃ for 24 h and nonprecooled donor cells. The cleavage rates, 4-cell development, and blastocyst development of reconstructed embryos were 72.48% (79/109), 53.16% (42/79), and 19.05% (8/42) in precooled group; 68.5% (211/308), 59.72% (126/211), and 17.46% (22/126) in nonprecooled group, respectively. Eighteen cloned 4-cell embryos derived from precooled donor cells were transferred and one cloned kid was born. Eighty-four cloned 4-cell embryos derived from nonprecooled donor cells were transferred and no offspring were produced. Of 18 cloned morale from nonprecooled donor cells transferred, one kid was born. The results of microsatellite DNA analyses indicated that the two cloned kids were from the same donor fibroblast cell line derived from an adult goat ear skin.     

The effect of electrical field strength on activation and development of cloned caprine embryos

The activation procedure used in nuclear transfer (NT) is one of the critical factors affecting the efficiency of animal cloning. The purpose of this study was to compare the effect of two electrical field strengths (EFS) for activation on the developmental competence of caprine NT embryos reconstructed from ear skin fibroblasts of adult Alpine does. The NT embryos were obtained by transfer of the quiescent fibroblasts at the fourth passage into the enucleated metaphase II (M II) oocytes. Four to five hours after electrical fusion, the NT-embryos were activated by EFS either at 1.67 or at 2.33 kV/cm and immediately incubated in 6-DMAP (2 mM) for 4 h. The cleavage rate of the NT-embryos activated with 2.33 kV/cm was greater than that activated with 1.67 kV/cm after in vitro culture for 18 h (65.6% versus 19.6%, p < 0.001). No pregnancy was found in 14 recipient does after transferring 51 NT embryos at 1-2 cell stages activated with 1.67 kV/cm. In contrast, two of the seven recipients were pregnant and gave birth to three kids after transferring 61 NT embryos at 1-2 cell stages activated by 2.33 kV/cm. The birth weights of three cloned kids were within the normal range of Alpine goats. However, one kid died 1h after birth while the remaining two are still healthy. DNA analysis by polymerase chain reaction (single-strand conformation polymorphism, SSCP) confirmed that the three kids were genetically identical to the nuclear donor.