Survival of DNA-injected cow embryos temporarily cultured in rabbit oviducts

Holstein or Angus cows were superovulated, inseminated with fresh bull semen, and necropsied about 12 h after estimated time of ovulation. Ova were centrifuged at 15,600 G for 3 to 8 min to reveal pronuclei. In Experiment 1, pronuclear bovine embryos were transferred to ligated or unligated oviducts of 1-d pseudopregnant rabbits for 7 d; 30 of 32 embryos were recovered from ligated oviducts but only 2 of 26 from oviducts and uterine horns of unligated oviducts. In Experiment 2, a Rous sarcoma virus-chloramphenicol acetyl transferase fusion gene was injected into one pronucleus of about half of 404 fertilized bovine ova, using a micromanipulator and interference contrast optics. Injected and noninjected embryos were then transferred to opposite ligated rabbit oviducts. Embryos were recovered after 7, 8 or 9 d. Of 120 centrifuged but ininjected embryos recovered from rabbit oviducts, 66 (55%) were in the morula to hatching blastocyst stage of development. Of 105 embryos centrifuged and injected with foreign DNA, 55 (52%) were in the morula to hatching blastocyst stage. In Experiment 3, centrifuged bovine embryos, noninjected or DNA-injected, were cultured in rabbit oviducts for 7 d then transferred nonsurgically to the uterus of recipient cows. Embryos were also flushed from superovulated cows 8 d after estrus and transferred directly to recipient cows. After 7 d, the uterus of recipient cows was flushed nonsurgically to recover embryos. The proportion of transferred embryos recovered with normally elongated trophoblastic membranes and the proportion of recipient cows with developing embryos were 14 of 25 DNA-injected embryos, 5 of 8 cows; 6 of 15 centrifuged but noninjected embryos, 4 of 6 cows; and 11 of 29 embryos transferred directly, 5 of 8 cows. Results indicate that bovine embryos can be cultured in rabbit oviducts and survive after transfer to cow uteri and that injection of foreign DNA may not increase embryonic loss within the first 2 wk after injection.     

Development of porcine ova that were centrifuged to permit visualization of pronuclei and nuclei

The obscured pronuclei or nuclei in living one- and two-celled pig ova were revealed after centrifugation for 3 min at 15,000 X g. To determine viability of centrifuged ova, one- and two-celled pig ova were collected from superovulated gilts; half of the ova were centrifuged and all ova were transferred into recipient gilts. Prior to transfer all embryos were stained with tetramethylrhodamine isothiocyanate (TRITC) to distinguish the experimental embryos from the recipients's own ova. Centrifuged ova were transferred into one oviduct of recipient gilts and uncentrifuged ova were deposited into the opposite oviduct. Embryos were recovered 4 days after transfer and were stained with lacmoid or Hoechst 33342 to assess their stage of development. Centrifugation had no detectable influence on survival of the recovered embryos to 4 days. Centrifugation is a simple, reliable method for revealing pronuclei and nuclei of one- and two-celled pig ova and apparently does not alter subsequent preimplantation development.     

The efficiency of production, centrifugation, microinjection and transfer of one- and two-cell bovine ova in a gene transfer program

Forty crossbred beef heifers were superovulated with 2000 IU pregnant mare serum gonadotropin (PMSG) and mated twice by natural service during estrus. Ovulations were counted and ova were recovered during mid-ventral laparotomy between 44 and 54 h after the onset of estrus. The overall donor ovulation rate (M+/-SEM) was 15.2+/-1.3. There was a positive association between ovulation rate and the number of ova recovered (P<0.001), and between ovulation rate and the incidence of ova advanced beyond the two-cell stage of development (P<0.05). When grouped on the basis of superovulation response, the numbers (M+/-SEM) of recovered one-cell, two-cell and more advanced ova were 3.7+/-0.7, 1.0+/-0.3 and 0.5+/-0.3, respectively, for donors with up to 15 ovulations. The corresponding numbers for donors with more than 15 ovulations were 7.2+/-1.8, 6.0+/-1.3 and 2.8+/-1.2, respectively. Following centrifugation, pronuclei were visible in 68% of one-cell ova, and nuclei were visible in 80% of two-cell ova. Approximately 20% of ova were destroyed during DNA microinjection. A total of 66 centrifuged and DNA-injected ova were transferred to the oviducts of 26 crossbred beef heifers, each receiving two, three or four ova. Echography at Day 55 confirmed that 14 (54%) heifers were pregnant with 26 (39%) fetuses. Eleven heifers were held to calve and produced 21 calves.     

Ovicidal effects of vitrification solution and the vitrification-warming cycle and establishment of the proportion of toxic effects on nuclei and cytoplasm of mouse zygotes

The present study was conducted to compare the resistance of the nuclei with that of the cytoplasm of mouse zygotes to damage during the vitrification-warming cycle using the technique of pronuclear transplantation. Zygotes were collected from the oviduct of superovulated F1 female mice mated with males of the same strain. They were cryopreserved by the vitrification method. After being diluted with glycerol-sucrose PB1 solution, 86% of the recovered zygotes were morphologically normal and 80% of them developed to the two-cell stage in vitro, but the proportion of zygotes which developed to blastocysts was only 27%. When zygotes were exposed to VS1 solution in the same manner as above without cooling, 61% of them developed to blastocysts. In order to examine the source of injury during vitrification, the pronuclei of vitrified zygotes were transferred into enucleated fresh zygotes and vice versa. The developmental rate of blastocysts from vitrified zygotes that were enucleated and fused with pronuclei from fresh zygotes was significantly higher than that of zygotes reconstituted reversely. These findings suggest that nuclei are apparently damaged more than cytoplasm by the vitrification-warming cycle and the toxicity of VS1 solution.     

Effects of superovulation, culture and microinjection on development of rabbit embryos in vitro

Factors influencing the developmental potential of cultured rabbit zygotes and their ability to incorporate and integrate the WAP-hPC (human protein C) gene were investigated. Rabbit zygotes (n = 1053) were recovered from both superovulated and nontreated New Zealand White females. The hormonal treatment of rabbit donors resulted in a doubling of the number of recovered ova per donor when compared with the nontreated group (18 vs 9 ova). However, the quality of recovered zygotes (presence of both pronuclei) was significantly better in the nontreated group (99 vs 88%, Experiment 1). The effect of various culture media on the development of rabbit zygotes in vitro was evaluated after incubation under CO2-free conditions (Experiment 2). In serum-free, growth factor-supplemented medium (BSEITS, DME/F12, 1.5% BSA, EGF, insulin, transferrin and sodium selenite) the percentage of morula/blastocyst stage embryos was significantly higher (88%) than in DME/FCS, (DME/F12, 10% fetal calf serum, 59%) or the control group (DME/F12, 1.5% BSA, 25%). In Experiment 3, zygotes were microinjected with the WAP-hPC gene and were examined after 72 h of culture. Zygote cleavage and the percentage of morula/blastocyst stage intact embryos were higher (79 and 58%, respectively) than in microinjected embryos (31.0 and 21.5%, respectively). Summarized data of the PCR assay of microinjected zygotes demonstrated positive signals for the integration of the WAP-hPC gene in 6.6% (34 of 515) of all the microinjected zygotes.     

Effect of pronuclear DNA microinjection on the development of porcine ova in utero

The objective of this study was to assess the effect of various aspects of pronuclear DNA microinjection on the early development of porcine ova in utero. Estrus was synchronized and superovulation was achieved in sexually mature gilts by the administration of allyl trenbolone, PMSG and hCG. Donor gilts were bred at 12 and 24 h after the onset of estrus. Ova were recovered between 60 and 62 h after the administration of hCG. One-cell ova that exhibited pronuclei after centrifugation were randomly allocated in equal numbers from each donor across one of two pairs of treatments: micro-DNA (ova were injected with two gene constructs that code for the human complement regulatory proteins decay accelerating factor and membrane cofactor protein) and control (ova were centrifuged only) or micro-buffer (ova were injected with buffer only) and pierced (a pipette was inserted into one pronucleus). Ova were transferred by treatment pairs to recipients. Treatments were segregated by oviduct. Ova were recovered after 120 h in utero, fixed and stained with 1% orcein. The proportion of ova that possessed > or = 80 nuclei, the mean number of nuclei present and proportion of ova that formed blastocysts were all significantly (P<0.05) greater for control and pierced ova than for micro-DNA and micro-buffer ova. No difference in these parameters was observed between micro-DNA and micro-buffer ova. These results demonstrate that pronuclear microinjection of a buffer alone can adversely affect the early development of porcine ova in utero.     

Method for producing transgenic bovine embryos from in vitro matured and fertilized oocytes

Microinjection and in vitro culture procedures were developed to produce transgenic bovine embryos after in vitro fertilization of in vitro matured oocytes. In Experiment I, zygotes were subjected to pronuclear microinjection of DNA 18 or 24 h following addition of spermatozoa to oocytes. Microinjections were performed in either Hepes-buffered TCM-199 or modified Dulbecco's phosphate-buffered saline without glucose. Viability of embryos was similar at both injection times and for both media, as determined by morphological evaluation after culturing embryos in vitro for 10 d. In Experiment II, microinjected embryos were cultured 1) in rabbit oviducts, 2) in vitro in a 5% CO(2) in air, or 3) in a 5% CO(2) / 5% O(2) / 90% N(2) incubator. There were no significant differences between the 2 in vitro culture environments. The in vitro culture systems supported development of embryos significantly better than the rabbit oviducts; 33% of cleaved ova developed to blastocysts in vitro vs 10% in vivo; 98% of transferred ova were recovered from the rabbit oviducts. From both experiments, 6 of 92 blastocysts were positive for the microinjected DNA as determined by a polymerase chain reaction followed by gel electrophoresis.     

Preimplantation development of rabbit embryos after transfer of embryonic nuclei into different cytoplasmic environment

The development of nuclear-transfer oocytes and zygotes was tested in the rabbit. Metaphase II oocytes and zygotes in the early pronuclear stage were treated with a cytoskeletal inhibitor (cytochalasin D), enucleated, and subsequently fused either with single blastomeres from eight- and 16-cell stages (oocytes and zygotes) or with pronuclei-containing karyoplasts (zygotes only). Also, nonenucleated zygotes were fused with 1/8 blastomeres. Fusion was performed by means of an electric field. Development of reconstituted embryos was monitored mainly in vitro, but a certain number of embryos developed from oocytes and zygotes receiving nuclei from eight-cell stages were also transferred into pseudopregnant does. Development of nuclear-transfer oocytes was distinctly better than that of nuclear-transfer zygotes, since 16.9% and 9.5% oocytes vs. 8.1% and 3.7% zygotes carrying eight- and 16-cell nuclei, respectively, developed to the blastocyst stage. Two advanced but already dead fetuses were found after transfer of 27 four-cell embryos obtained after fusion of oocytes with 1/8 blastomeres. No implantations were observed after transfer of 25 four-cell embryos developed from enucleated zygotes receiving eight-cell nuclei. These findings indicate that, in the rabbit, some nuclei from 16-cell embryos are still capable of promoting at least preimplantation development. Comparison between the developmental abilities of oocyte- and zygote-derived nuclear-transfer embryos also suggests that the cytoplasmic environment of recipient cell is more crucial for the development of reconstituted embryos than the stage of introduced nuclei (at least up to the 16-cell stage). The majority of pronuclear exchange embryos (69.9%) and 40% of nonenucleated zygotes receiving eight-cell nuclei were able to develop to the blastocyst stage. This latter observation indicates, similarly as with mouse, a supporting role of residual pronuclei for participation of an eight-cell nucleus in the development of reconstituted zygotes.     

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.     

激肽释放酶转基因大鼠模型的建立

目的建立KLK1转基因大鼠模型。方法腹腔注射PMSG-HCG（150-150IU/kg）超排不同周龄（4-8周）SD大鼠比较超排效果的差异,以可用于注射胚胎数作为评判标准确定最佳超排周龄。构建pBC-klk1转基因构件,经酶切、纯化后通过显微注射方法导入SD大鼠受精卵原核并移植到同期受孕的SD受体母鼠输卵管内。出生后仔鼠用PCR和Southern方法检测鼠尾DNA鉴定基因型,通过RT-PCR和免疫组化方法检测klk1基因表达。结果4-8周SD大鼠超排后分别获得受精卵14±14.8、30±15.2、13.3±13.7、13±14.7、8±5.7,5周龄大鼠超排效果最好,与其他周龄大鼠相比有显差异,P〈0.05;显微注射1538枚卵,移植685（44.53%）枚卵于31只受体输卵管中,12（12/31,38.7%）只怀孕,共产仔62只,经PCR检测获得6只阳性鼠,Southern检测3只阳性。对Southern检测阳性转基因大鼠子代进行RT-PCR检测和免疫组化分析证明klk1基因在肾脏、胰腺和乳腺内表达。结论成功建立klk1基因表达的转基因大鼠模型,该模型是高血压病研究的理想动物模型。     

Development and survival after transfer of cow embryos cultured from 1-2-cells to morulae or blastocysts in rabbit oviducts or in a simple medium with bovine oviduct epithelial cells

This study compares development of bovine 1-2-cell embryos in bovine oviduct epithelial cell co-culture (Group EC) with a glucose- and serum-free simple medium (CZB), or after surgical transfer to ligated oviducts of rabbits (Group RO). Embryos were surgically collected from superovulated donor cows 40-48 h after the beginning of oestrus and randomly distributed between the two groups. Embryos were cultured or incubated for 5 days. In Exp. 1, embryo quality scores and total numbers of cells in the two groups were compared. In Exp. 2, pairs of similarly treated morulae were transferred to each of 10 or 12 recipients in the Groups RO and EC, respectively. Total cell counts per embryo in both groups averaged 52 (P greater than 0.05), and the in-vitro culture system was equivalent to the rabbit oviducts in promoting embryo development for all characteristics measured. Embryo survival, as determined by ultrasound between Days 39 and 43 after oestrus, in 13 ideal recipients was 57% for embryos in Group EC and 58% for embryos Group RO. None of the 9 less desirable recipients was pregnant for either group. These results establish that cattle zygotes can develop to morulae in culture with bovine oviduct epithelial cells in a simple medium and can produce normal pregnancy rates.     

Visualization of pronuclei in living bovine zygotes

Bovine embryos were surgically collected from the oviducts of superovulated crossbred heifers 48 h postonset of estrus. The one-celled ova were treated with 4'-6'-diamidino-2-phenylindole (DAPI) and observed under ultraviolet light by fluorescence microscopy. Both male and female pronuclei were visualized, identified and subjected to micromanipulation.     

Developmental capacity of rat embryos produced by in vivo or in vitro fertilization

This work compares the ability of rat zygotes fertilized in vitro or in vivo to develop into viable embryos. All oocytes were from adult cyclic females. After the first cleavage, the zygotes were transferred to oviducts of pseudopregnant recipients. Their fate was examined on day 13 at laparotomy and again on day 20. Ninety-five of 146 in vivo fertilized zygotes developed into normal sized 13-day fetuses and 72 (55%) to apparently normal near-term fetuses. Forty-six of 135 in vitro fertilized zygotes developed up to day 13, and 30 (24%) developed to term. It appears that the probability that in vitro fertilized rat zygotes will develop into viable embryos is about half the chance of in vivo fertilized zygotes. Since the two types of zygotes were morphologically identical, the morphological appearance of the two-cell stage is not an adequate criterion for judging developmental potential.     

Developmental potential of early bovine zygotes submitted to centrifugation and microinjection following in vitro maturation of oocytes

This study was designed to investigate the potential use of in vitro matured, in vitro fertilized bovine zygotes for producing transgenic calves by microinjection of foreign DNA. In Experiment 1, the effect of centrifugation (4 min, 15,000 x g, 20 degrees C) on in vitro derived bovine zygotes was evaluated. In vitro development from 2 to 8 cells was not affected (80 vs 78%) when control zygotes (n = 211) were compared with zygotes treated (n = 210) 18 h post insemination. In Experiment 2, the influence of the centrifugation alone on the developmental potential of embryos was evaluated in rabbit oviducts for 120 h. The percentage of control and treated zygotes that developed to 1, 2 to 8, 8 to 32 and more than 32 cells were 7, 54, 10 and 10% vs 7, 40, 11 and 10%, respectively. In Experiment 3, the effect of pronuclear injection with plasmid containing CRF (corticotropin releasing factor) gene or pOCAT 330 Delta1 plasmids; 2 mug/ml in Tris 10 mM, EDTA 0.2 mM, 18 to 20 h post insemination was evaluated by in vivo development in the rabbit oviduct. The embryos submitted only to centrifugation and vortexing resulted in a morula-blastocyst (> 32 cells) rate of 25% (n = 226) compared with the injected zygotes of which only 5% (n = 206) achieved the same stage. We conclude that in vitro produced bovine zygotes have a reduced developmental potential following microinjection, and this effect is not due to the centrifugation process.     

Pronuclear visibility, development and transgene expression in IVM/IVF-derived porcine embryos

A total of 1550 zygotes was used to assess the timing of pronuclear visibility, embryo development following DNA microinjection, and transgene expression in IVM/IVF-generated porcine embryos. After centrifugation, pronuclei could be seen in 61.6% of zygotes. In 55.3% of these only 1 pronucleus was visible. Pronuclear visibility was highest at 20 h post-insemination. Zygotes were microinjected with 1 of 2 LacZ gene constructs driven by either the SV40 early promoter (pSVON) or the human cytoplasmic beta actin promoter (pbActinLacZ). Development and transgene expression were assessed after either 48 h or 7 d in culture. After 48 h, significantly more zygotes with a single visible pronucleus developed to the 8-cell stage than zygotes in which no pronucleus had been seen (43.0 vs 24.8%), while those with 2 pronuclei were intermediate (31.4%). After 7 d, no difference in development to the morula stage was observed between noninjected control embryos (25.5%) and embryos with 1 (21.0%) or 2 pronuclei (22.5%); however, the proportion of embryos reaching the morula stage in the nonpronuclear group was significantly reduced (9.1%). After 48 h in culture, transgene expression was significantly higher in embryos with 2 pronuclei at the time of injection than in those with 1 (36.4 vs 17.9%). After 7 d in culture, 41.5% of morulae derived from zygotes with 2 pronuclei and 29.97% of thsoe derived from zygotes with 1 pronucleus showed signs of transgene expression. At this stage, significantly more morulae expressed the pbActinLacZ than the pSVON transgene (43.8 vs 25.8%). More than 80% of putative transgenic morulae or blastocysts showed evidence of mosaicism. These results demonstrate that IVM/IVF porcine embryos are able to develop in culture and express a microinjected transgene.     

Effect of morphological properties of transferred embryonic stages on tubal migration Implications for in vivo culture in the bovine oviduct

In cattle, there is no practical method, which allows tubal transfer of pre-implantation embryos for routine in vivo culture as it has been established in sheep. The aim of our study was to perform tubal transfer by transvaginal endoscopy in synchronized heifers, in order to expose embryos at various embryonic stages to the physiological mechanisms of migration in the non-ligated oviducts. Various embryonic stages were transferred by transvaginal endoscopy into the oviducts of temporary recipients and were recovered on Day 7. The transfer of embryos in hyaluronate containing medium ("Hyaluronan"), zygotes stripped of cumulus ("Denuded Zygotes"), embryos embedded in cumulus ("Zygotes with Cumulus"), matured oocytes with capacitated spermatozoa ("GIFT") or embryos embedded in Na alginate ("Alginate") led to increasing recovery rates (13, 30, 56, 63 and 71%, respectively). However, the developmental rate on Day 7 was adversely affected (16, 11, 8, 16 and 8%), whereas the blastocyst rate on Day 8 showed more balanced results (17, 14, 18, 21 and 11%). Our data demonstrate that the structural properties of transferred embryos affect tubal migration and are crucial for subsequent in vivo culture. Embryos enclosed in cumulus cells or alginate synchronize more successfully with the oviductal transport systems than denuded stages or embryos in hyaluronate containing medium.     

Ultrastructure of in vivo fertilization in the goat

In vivo fertilization of goat eggs has been studied by electron microscopy. Eggs were recovered from superovulated or natural cyclic goats, 32 to 52 hours after the onset of oestrus; only eggs recovered between 46 and 52 hours were fertilized. Spermatozoa penetrated the zona pellucida tangentially leaving vesiculated products of the acrosome reaction at the zona surface. As sperm penetrated into the ooplasm, the second meiotic division completed and cortical granule exocytosis occurred. However a few unreacted cortical granules usually remained in the cortex of the two fertilized eggs, adjacent to the plasma membrane. After swelling the two pronuclei presented similar ultrastructural morphology: they contained small, compact, agranular nucleoli and unevenly distributed chromatin. The cytoplasm in close vicinity to the apposed pronuclei contained large stacks of annulate lamellae, smooth endoplasmic reticulum, prominent Golgi complexes, as well as dense areas of unidentified material. The abundance of cytoplasmic organelles near the pronuclei might be the expression of intensive metabolic activity. Conversely, in the cortex of fertilized ova several large organelles-free cytoplasmic areas were randomly distributed.     

血红素加氧酶-1显性负性突变体转基因小鼠模型的建立与鉴定

目的建立血红素加氧酶-1(heme oxygenase-1,HO-1)显性负性突变体G143H转基因小鼠模型。方法通过SalI/DraI酶切pCAGGHO-1G143H转基因表达载体,纯化回收HO-1G143H表达盒片段;通过显微注射把表达目的基因的DNA片段导入FVB小鼠受精卵原核,并移植给同期发情的假孕受体母鼠,获得子代小鼠;用PCR对子代鼠尾DNA进行鉴定,并用Southern blot对结果做进一步验证;通过RT-PCR、免疫组化和Western blot方法检测HO-1基因的表达。结果表达盒回收片段正确;假孕鼠出生的17只子代小鼠共有3只阳性,均为雄性;RT-PCR、免疫组化和Western blot结果表明,阳性小鼠体内的HO-1 mRNA与蛋白表达水平增高。结论成功建立HO-1显性负性突变体G143H表达的转基因小鼠,该模型为研究HO-1在体内的作用机制奠定了基础。     

Superovulatory responses of Holstein cows

Approximately 1000 registered cows and heifers were superovulated one to 10 times. Nonsurgical embryo recoveries were performed on all donors which exhibited estrus. Healthy donors produced more total ova and cleaving embryos and had a higher ovum recovery rate, fertilization rate and pregnancy rate from embryos transferred than did cows classified as infertile. While ovum number was not affected during 10 repeated superovulations, fertilization rate and embryo number decreased. The number of ova recovered from healthy cows was affected by season, and from infertile cows by the day of the estrous cycle on which FSH was started and by the number of days since calving. More ova were recovered from infertile cows synchronized with prostaglandins prior to superovulation than following a natural estrous cycle. The number of embryos recovered from infertile cows was affected by age and from healthy cows by daily milk production. Fertilization rates in both healthy and infertile cows were affected by age, time since calving, daily milk production, day of cycle FSH was injected and season. There was no effect of the day of recovery on the number of ova or embryos recovered from healthy or infertile cows.     

In vivo development of microinjected embryos from superovulated prepuberal slaughter lambs

This study was performed to investigate the developmental potential of microinjected embryos recovered from superovulated prepuberal lambs. Fifty-nine mixed-bred lambs (about 3 mo old) were superovulated either with 18 mg FSH-P with (Group FSH/+S) or without (Group FSH/-S) progestagen treatment, or with 10 mL Ovagene following progestagen treatment (Group OVA/+S). All animals received hCG to induce ovulation. Ovulation rates and the number of ova recovered per animal for the different groups were 8.7 and 4.7 (55%, FSH/+S); 7.3 and 3.2 (42%, FSH/-S); and 6.4 and 4.0 (65%, OVA/+S), respectively. No significant differences were seen in the ovulation and the recovery rates, but animals without progestagen treatment showed a significantly lower fertilization rate (44%) when compared with progestagen-treated groups (87%; P<0.001). Foreign DNA was microinjected into the pronuclei of fertilized ova (n = 155), which were transferred (n = 98) into synchronized recipient ewes (n = 21). Two animals were detected pregnant and both gave birth to a single lamb. Results of superovulation and embryo recovery from prepuberal lambs were promising, but the low rate of development to term indicates that ova recovered from prepuberal lambs have reduced developmental competence in vivo. Although 2 lambs were born, it seems that this is not a successful method for use in future gene transfer programs.