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Background: Epididymal sperm preservation is a simple conservation approach that can help prevent the loss of high genetic quality of farm animals. The chance of loss increases, especially during disease outbreaks or other interruptions to normal reproduction function., Aim: This study looked into the ability of preserved ram epididymal sperm to fertilize oocytes. Due to motility becoming an issue following sperm storage for fertilization, the sperm microinjection known as intracytoplasmic sperm injection approach was employed., Methods: The study was divided into two parts. First, involved the preservation of epididymal sperm at 5°C for 12 days. During preservation, sperm quality parameters namely motility, viability, intact membrane, acrosome, and Deoxyribonucleic acid (DNA) are evaluated every three days. For the fertility test in the second experiment, matured oocytes were injected with immotile sperm discovered in the last days of preservation. The presence of pronucleus development following in vitro culture is used as an indicator of sperm's ability to activate and fertilize oocytes., Results: All sperm quality parameters significantly ( p < 0.05) declined during preservation time. On day 12, motility was discovered to be 0%, but viable sperm, sperm with intact membrane, acrosome, and DNA remained at 41.86% ± 9.30%, 31.18% ± 5.15%, 21.88% ± 1.93%, and 33.35% ± 8.74%, respectively. On the fertility test, we inject immotile sperm from day 12 of preservation, which has the lowest motility found, into matured oocytes. Those sperms are able to activate (52.05% ± 7.15%) and fertilize (31.37% ± 1.75%) the injected oocytes, but their fertilizing ability is significantly lower ( p < 0.05) when compared to the sperm derived from the ejaculate., Conclusion: In this study, simple preservation of epididymal sperm reduces all sperm quality criteria, particularly motility. Using the microinjection approach preserved sperm which had no motility, still demonstrated its ability to activate and fertilize the oocytes. According to that, this study provides potential approaches and tools for using genetically superior animals that have lost their ability to execute regular fertilization, and also prolong reproduction function., Competing Interests: The authors declare that there is no conflict of interest in the current study.

Bone morphogenetic protein 15 (BMP15) is an X-linked gene encoding an oocyte secreted factor, which plays varied functions in the female fertility between mono-ovulatory and poly-ovulatory mammalian species. We previously found that knockout of BMP15 completely blocked porcine follicular development at preantral stages. However, the specific function of BMP15 on porcine oocytes in vitro maturation remains largely unknown. Here, we injected the pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complex into the cytoplasm of germinal vesicle stage porcine oocytes to disrupt BMP15. The ctRNP composed of Cas9 nuclease and crRNA-tracrRNA complex at 1.2/1 content ratio. The tested crRNA-tracrRNA complex concentration ranging from 50 to 200 ng/μL, all presented effective editing of BMP15 in porcine oocytes, and the 125 ng/μL crRNA-tracrRNA complex presented the highest editing efficiency (39.23 ± 3.33%). Surprisingly, we found approximately 95% edited oocytes presented monoallelic mutations, and only 5% edited oocytes harbored biallelic mutations. Interestingly, the coinjected two crRNAs guided the ctRNP complex to concurrently cut within a 10 bp window of the PAM (protospacer adjacent motif), resulting in a precise deletion within BMP15 in 85.9% edited oocytes, and additional deletion happened in 14.1% edited oocytes, which resulted in large fragment deletions in BMP15. Most deletions caused frameshift and introduced premature stop codon in BMP15, resulting in the disruption of BMP15 protein expression, which was confirmed by the Western blot analysis showing the reduced BMP15 protein expression in ctRNP injected oocytes. The disruption of BMP15 attenuated the activation of SMAD1/5/8 signaling, and impaired cumulus expansion of porcine cumulus cell-oocyte complexes (COCs). Our study proved that delivering CRISPR ctRNP into porcine oocytes by microinjection was able to edit BMP15 efficiently, providing a new strategy to investigate the functions of oocyte-specific secreted factors in oocyte in vitro maturation., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript or in the decision to publish the results., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Cytosine base editors (CBEs) and CRISPR/Cas9-mediated HDR method both have the ability to introduce nucleotide substitution into genomes, which exhibit great potential for improving economically important traits in livestock species. The FecG H mutation (g. C1184T, p. S395F) of growth differentiation factor 9 (GDF9) gene increases prolificacy in Cambridge sheep and Belclare sheep. In the present study, we aimed to compare the efficiency and precision of BE4-Gam and CRISPR/Cas9 systems on generating FecG H mutation in ovine genome. First, the microinjection of BE4-Gam mRNA had no adverse effects on development rate after cleavage, and the efficiencies of total mutants and targeted mutants were 8.9% and 7.1%, respectively. Then, the total mutation and targeted mutation rates were improved from 8.5% to 22.5% (p < .01), and 6.4% to 16.3%, respectively, by adjusting the injection time of BE4-Gam mRNA from 14 to 12 hr post-insemination (hpi). Furthermore, CRISPR/Cas9-mediated HDR method introduced the FecG H mutation at the efficiency of 16.1%, which was comparable to BE4-Gam system (16.3%). There was no bystander editing event happened in edited embryos caused by CRISPR/Cas9, but the bystander editing efficiency was as high as 15.0% in BE4-Gam-edited embryos. In summary, our findings demonstrated that CRISPR/Cas9-mediated HDR method was more accurate than BE4-Gam system in introducing FecG H into ovine genome, and highlight the potential of the former strategy to modify economically important trait-associated SNPs., (© 2022 Wiley-VCH GmbH.)

Limb girdle muscular dystrophy type R1 (LGMDR1) is an autosomal recessive myopathy described in humans resulting from a deficiency of calpain-3 protein (CAPN3). This disease lacks effective treatment and an appropriate model, so the generation of KO pigs by CRISPR-Cas9 offers a way to better understand disease ethology and to develop novel therapies. Microinjection is the main method described for gene editing by CRISPR-Cas9 in porcine embryo, but electroporation, which allows handling more embryos faster and easier, has also recently been reported. The objective of the current study was to optimize porcine oocyte electroporation to maximize embryo quality and mutation rate in order to efficiently generate LGMDR1 porcine models. We found that the efficiency of generating CAPN3 KO embryos was highest with 4 electroporation pulses and double sgRNA concentration than microinjection. Direct comparison between microinjection and electroporation demonstrated similar rates of embryo development and mutation parameters. The results of our study demonstrate that oocyte electroporation, an easier and faster method than microinjection, is comparable to standard approaches, paving the way for democratization of transgenesis in pigs., Competing Interests: Declaration of competing interest None of the authors have any conflict of interest to declare., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The ability to efficiently introduce site-specific genetic modifications to the mammalian genome has been dramatically improved with the use of the CRISPR/Cas9 system. CRISPR/Cas9 is a powerful tool used to generate genetic modifications by causing double-strand breaks (DSBs) in DNA. Artemis (ART; also known as DCLRE1C), is a nuclear protein and is essential for DSB end joining in DNA repair via the canonical non-homologous end joining (c-NHEJ) pathway. In this work, we tested whether ART deficiency affects DNA repair following CRISPR/Cas9 induced DSBs in somatic cells. We also demonstrated the effect of microinjection timing on embryo developmental ability and gene targeting efficiency of CRISPR/Cas9 system to disrupt the interleukin 2 receptor subunit gamma (IL2RG) locus using porcine in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) derived embryos. In comparison to non-injected controls, CRISPR/Cas9 injection of IVF derived zygotes at 4 h and 8 h after fertilization did not impact cleavage and blastocyst rate. Gene modification rate was observed to be higher, 53.3% (9/16) in blastocysts injected 4 h post-fertilization as compared to 11.1% (1/9) in blastocysts injected 8 h post-fertilization. Microinjection 8 h after chemical activation of SCNT derived embryos decreased blastocyst development rate compared to non-injected controls but showed a higher gene modification efficiency of 66.7% as compared to 25% in the 4 h post-activation injection group. Furthermore, we observed that male ART -/- and ART +/- porcine fetal fibroblast (pFF) cells showed lower modification rates (2.5% and 1.9%, respectively) as compared to the ART intact cell line (8.3%). Interestingly, the female ART -/- and ART +/- pFF cells had modification rates (4.2% and 10.1%, respectively) similar to those seen in the ART intact cells. This study demonstrates the complex effect of various parameters such as microinjection timing and ART deficiency on gene editing efficiency in in vitro derived porcine embryos., Competing Interests: Declaration of competing interest None., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Microinjection of exogenous DNA into the cytoplasm of matured oocytes or zygotes is a promising technique to generate transgenic animals. However, the data about the microinjection time and procedure in sheep are limited and have not treated in detail. To obtain more in-depth information, the Sarda sheep oocytes from abattoir-derived ovaries were subjected to IVM and IVF. Then, the GFP plasmid as a reporter gene was injected into the cytoplasm of MII oocytes (n: 95) and zygotes at different post-insemination intervals (6-8 hpi, n: 120; 8-10 hpi, n: 122; 10-12 hpi, n: 110 and 12-14 hpi, n: 96). There were no significant differences in the cleavage rates between the groups. However, blastocyst rate of injected zygotes at all-time intervals was significantly lower than injected MII oocytes and control group (p < 0.05). Interestingly, the proportion of GFP-positive embryos was higher at 8-10 hpi compared with other injected groups (4 % versus 0 %, p  < 0.01). Among these, the proportion of mosaic embryos was high and two of those embryos developed to the blastocyst stage. In conclusion, we settled on the cytoplasmic microinjection of GFP plasmid at 8-10 hpi as an optimized time point for the production of transgenic sheep and subsequent experiments., (© 2020 Wiley-VCH GmbH.)

Background: Post-clipping alopecia often has a clinically poor response to therapy and prolonged alopecia is a source of anxiety for some owners. In humans and dogs, superficial microtrauma via a microneedling (MN) device induces mechanical stimulation of the hair follicle with resultant hair regrowth. Human studies suggest that concurrent application of platelet-rich plasma (PRP) with MN induces more rapid regrowth of better-quality hair than microneedling alone., Hypothesis: Microneedling with PRP will induce more rapid regrowth of better quality hair., Animals: Four unrelated client-owned dogs diagnosed with post-clipping alopecia., Methods and Materials: This was a prospective study. The affected site was divided in half, with the first half treated with MN alone and the second half treated with MN + PRP. Hair regrowth was assessed by clinician and owner using a hair growth assessment scale (HGAS) at one, three, six and 12 months., Results: At three months, all dogs had improved and three exhibited greater hair regrowth on the MN + PRP side. A similar response was noted bilaterally in three dogs, which had improved by 76-100% at six months and remained unchanged at 12 months. One dog improved by < 26% at six months, but had> 50% re-growth by 12 months. The small sample size precluded statistical analysis., Conclusions and Clinical Importance: In dogs with post-clipping alopecia, MN + PRP appeared to induce more rapid hair regrowth than MN; however, overall results were visibly equivalent by six months regardless of method. Both MN and MN + PRP proved successful for treating post-clipping alopecia., (© 2019 ESVD and ACVD.)

Important genomic imprinting changes usually occur following the in vitro production (IVP) of bovine embryos, especially in the imprinting pattern of components of the IGF system. This study aimed to evaluate the effects of a transient episomal overexpression of the IGF2 gene in bovine IVP embryos following embryo cytoplasmic microinjection (CMI) at the 1-cell stage on embryo survival, early and late developmental kinetics and morphological quality up to Day 7 of development. Selected cumulus-oocyte complexes (COCs) were matured and fertilized in vitro and subsequently segregated into six experimental groups: non-CMI control group and five CMI groups at increasing doses (0, 10, 20, 40 and 80 ng/μl) of a GFP vector built for the episomal expression of bovine IGF2. Zygote CMI was effective in delivering the expression vector into the ooplasm, irrespective of the groups, with 58% of positive GFP fluorescence in Day 7 blastocysts. Considering developmental rates and late embryo kinetics, the 10-ng/μl CMI vector dose promoted a lower blastocyst rate (10.4%), but for blastocysts at more advanced stages of development (93.0% blastocysts and expanded blastocysts), and higher number of cells (116.0 ± 3.0) than non-CMI controls (23.3%, 75.0% and 75.0 ± 6.8 were obtained, respectively). In conclusion, CMI at the 1-cell stage did not compromise subsequent in vitro development of surviving embryos, with the 10-ng/μl group demonstrating a possible growth-promoting effect of the IGF2 gene on embryo development, from the 1-cell to the blastocyst stage., (© 2020 Blackwell Verlag GmbH.)

The next generation of new genetically engineered rat models by microinjection is described. Genome editors such as CRISPR/Cas9 have greatly increased the efficiency with which the rat genome can be modified to generate research models for biomedical research. Pronuclear microinjection of transgene DNA into rat zygotes results in random multicopy transgene integration events that use exogenous promoters to drive expression. Best practices in transgenic animal design indicate the use of precise single copy transgene integration in the genome. This ideal can be achieved by repair of CRISPR/Cas9 chromosome breaks by homology directed repair. The most effective way to achieve this type of transgenic rat model is to deliver genome modification reagents to rat zygotes by pronuclear microinjection. The keys to success in this process are to obtain fertilized eggs (zygotes) from the rat strain of choice, to purify the microinjection reagents, to deliver the reagents to the eggs by pronuclear microinjection, to use the surgical transfer of microinjected eggs to pseudopregnant rats to obtain G0 founder animals that carry the novel genetic modification. Ultimately the success of new rat models is measured by changes in gene expression as in the expression of a new reporter protein such as eGFP, Cre recombinase, or other protein of interest.

Here we report in vitro and term development of sheep embryos after the inner cell mass (ICM) from one set of sheep blastocysts were injected into the trophoblast vesicles of another set. We also observed successful in vitro development of chimeric blastocysts made from sheep trophoblast vesicles injected with bovine ICM. First, we dissected ICMs from 35 sheep blastocysts using a stainless steel microblade and injected them into 29 re-expanded sheep trophoblastic vesicles. Of the 25 successfully micromanipulated trophoblastic vesicles, 15 (51.7%) re-expanded normally and showed proper ICM integration. The seven most well reconstructed embryos were transferred for development to term. Three ewes receiving manipulated blastocysts were pregnant at day 45 (42.8%), and all delivered normal offspring (singletons, two females and one male, average weight: 3.54 ± 0.358 kg). Next, we monitored in vitro development of sheep trophoblasts injected with bovine ICMs. Of 17 injected trophoblastic vesicles, 10 (58.8%) re-expanded after 4 h in culture, and four (40%) exhibited integrated bovine ICM. Our results indicate that ICM/trophoblast exchange is feasible, allowing full term development with satisfactory lambing rate. Therefore, ICM exchange is a promising approach for endangered species conservation.

Recently, successful one-step genome editing by microinjection of CRISPR/Cas9-related mRNA components into the porcine zygote has been described. Given the relatively long gestational period and the high cost of housing swine, the establishment of an effective microinjection-based porcine genome editing method is urgently required. Previously, we have attempted to disrupt a gene encoding α-1,3-galactosyltransferase (GGTA1), which synthesizes the α-Gal epitope, by microinjecting CRISPR/Cas9-related nucleic acids and enhanced green fluorescent protein (EGFP) mRNA into porcine oocytes immediately after electrical activation. We found that genome editing was indeed induced, although the resulting blastocysts were mosaic and the frequency of modified cells appeared to be low (50%). To improve genome editing efficiency in porcine oocytes, cytoplasmic injection was performed 6 h after electrical activation, a stage wherein the pronucleus is formed. The developing blastocysts exhibited higher levels of EGFP. Furthermore, the T7 endonuclease 1 assay and subsequent sequencing demonstrated that these embryos exhibited increased genome editing efficiencies (69%), although a high degree of mosaicism for the induced mutation was still observed. Single blastocyst-based cytochemical staining with fluorescently labeled isolectin BS-I-B 4 also confirmed this mosaicism. Thus, the development of a technique that avoids or reduces such mosaicism would be a key factor for efficient knock out piglet production via microinjection., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Microinjection is a widely used technique to inject defined volumes and concentrations of substances and explore their physiological function in vivo. The technique has been particularly successful with zebrafish embryos; however, the injection equipment can be relatively expensive and therefore available only to well-funded laboratories. In this study, a simple, cheap, easy-to-assemble, and easy-to-use setup with a straightforward, accurate, and efficacious calibration method is introduced. The accuracy of this calibration method was tested by comparing with the results of calibration methods that are currently used in high-cost systems. Injection success with this low-cost system was verified based on the presence of injected dyes in zebrafish embryos, the absence of any significant morphological and behavioral differences between 3,4,-dichloroaniline-treated and untreated embryos, and larval viability.

Background: We describe the fabrication and performance of a chronic in situ coil system designed to allow focal brain stimulation in rats while acquiring functional MRI data., New Method: An implantable receive-only surface radiofrequency coil (iCoil) was designed to be fitted subcutaneously, directly onto to the rat skull surface during the intracerebral cannulation procedure. The coil is fixed in place using acrylic dental cement anchored to four screws threaded into the skull. To demonstrate the use of this coil system in situ, whole-brain functional MRI scans were acquired during various stimuli, including intracranial microinfusions of bicuculline and morphine in the prefrontal cortex and ventral tegmental area, respectively., Results/comparison to Other Methods: SNR performance of the iCoil was superior to three commercially-available coils, in some instances by a factor of two. Widespread BOLD activation was observed in response to bicuculline and morphine microinfusions., Conclusion: A new approach was demonstrated for high-SNR MR imaging of the brain in rats with intracranial implants using an implantable surface coil. This approach enables mapping the functional response to highly targeted stimuli such as intracranial microinfusions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The zebrafish model has been used to determine the role of vertebrate innate immunity during bacterial infections. Here, we compare the in vivo immune response induced by GFP-tagged Salmonella Typhimurium inoculated by immersion and microinjection in transgenic zebrafish larvae. Our novel infection protocols in zebrafish allow live-cell imaging of Salmonella colonization., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Microinjection of zebrafish larvae is an essential technique for delivery of treatments, dyes, microbes, and xenotransplantation into various tissues. Although a number of casts are available to orient embryos at the single-cell stage, no device has been specifically designed to position hatching-stage larvae for microinjection of different tissues. In this study, we present a reusable silicone device consisting of arrayed microstructures, designed to immobilize 2 days postfertilization larvae in lateral, ventral, and dorsal orientations, while providing maximal access to target sites for microinjection. Injection of rhodamine dextran was used to demonstrate the utility of this device for precise microinjection of multiple anatomical targets.

The development of genome editing technology has allowed gene disruptions to be achieved in various animal species and has been beneficial to many mammals. Gene disruption using pluripotent stem cells is difficult to achieve in rabbits, but thanks to advances in genome editing technology, a number of gene disruptions have been conducted. This paper describes a simple and easy method for carrying out gene disruptions in rabbits using CRISPR/Cas9 in which the gene to be disrupted is marked, the presence or absence of off-target candidates is checked, and a plasmid allowing simultaneous expression of Cas9 and sgRNA is constructed. Next, the cleaving activity of candidate sequences is investigated, and assessments are carried out to determine whether the target sequences can be cut. Female rabbits subjected to superovulation treatment are mated with male rabbits and fertilized eggs are collected, and then pronuclear injection of plasmid DNA is performed. The next day, the two-cell stage embryos are transplanted into pseudopregnant rabbits, and offspring are born within approximately 29-30 days. The genomic DNA of the offspring is then examined to check what types of genetic modifications have occurred. With the advent of CRISPR/Cas9, the accessibility of gene disruptions in rabbits has improved remarkably. This paper summarizes specifically how to carry out gene disruptions in rabbits.

Live-attenuated oral rotavirus (RV) vaccines have lower efficacy in low income countries, and additionally are associated with a rare but severe adverse event, intussusception. We have been pursuing the development of an inactivated rotavirus vaccine (IRV) using the human rotavirus strain CDC-9 (G1P[8]) through parenteral immunization and previously demonstrated dose sparing and enhanced immunogenicity of intradermal (ID) unadjuvanted IRV using a coated microneedle patch in comparison with intramuscular (IM) administration in mice. The aim of this study was to evaluate the immune response and protection against RV infection and diarrhea conferred by the administration of the ID unadjuvanted IRV using the microneedle device MicronJet600® in neonatal gnotobiotic (Gn) piglets challenged with virulent Wa G1P[8] human RV. Three doses of 5 μg IRV when administered intradermally and 5 μg IRV formulated with aluminum hydroxide [Al(OH)3] when administered intramuscularly induced comparable rotavirus-specific antibody titers of IgA, IgG, IgG avidity index and neutralizing activity in sera of neonatal piglets. Both IRV vaccination regimens protected against RV antigen shedding in stools, and reduced the cumulative diarrhea scores in the piglets. This study demonstrated that the ID and IM administrations of IRV are immunogenic and protective against RV-induced diarrhea in neonatal piglets. Our findings highlight the potential value of an adjuvant sparing effect of the IRV ID delivery route., Competing Interests: The support in the form of salaries does not alter our adherence to PLOS ONE policies on sharing data and materials.

Junction-mediating and regulatory protein (JMY) is a regulator of both transcription and actin filament assembly. JMY is a critical nucleation-promoting factor (NPF); however, its role in the development of mammalian embryos is poorly understood. In the current study, we investigated the functional roles of the NPF JMY in porcine embryos. Porcine embryos expressed JMY mRNA and protein, and JMY protein moved from the cytoplasm to the nucleus at later embryonic developmental stages. Knockdown of JMY by RNA interference markedly decreased the rate of blastocyst development, validating its role in the development of porcine embryos. Furthermore, injection of JMY dsRNA also impaired actin and Arp2 expression, and co-injection of actin and Arp2 mRNA partially rescued blastocyst development. Taken together, our results show that the NPF JMY is involved in the development of porcine embryos by regulating the NPF-Arp2-actin pathway.

Krüppel-like protein Gli-similar 1 (GLIS1) is known as a direct reprogramming factor for the generation of induced pluripotent stem cells. The objective of this study was to investigate the role of GLIS1 in the preimplantation development of bovine embryos. GLIS1 transcripts in in vitro-matured oocytes and 1-cell to 4-cell stage embryos were detected, but they were either absent or at trace levels at the 8-cell to blastocyst stages. We attempted GLIS1 downregulation of bovine early embryos by RNA interference and evaluated developmental competency and gene transcripts, which are involved in zygotic gene activation (ZGA) in GLIS1-downregulated embryos. Injection of specific siRNA resulted in a distinct decrease in GLIS1 transcript in bovine embryos at the 4-cell stage. Although the bovine embryos injected with GLIS1-siRNA could develop to the 16-cell stage, these embryos had difficulty in developing beyond the 32-cell stage. Gene transcripts of PDHA1 and HSPA8, which are transcribed after ZGA, showed lower level in GLIS1 downregulated embryos. It is possible that GLIS1-downregulated embryos fail to initiate ZGA. Our results indicated that GLIS1 is an important factor for the preimplantation development of bovine embryos.

The process of transgenesis involves the introduction of a foreign gene, the transgene, into the genome of an animal. Gene transfer by pronuclear microinjection (PNI) is the predominant method used to produce transgenic animals. However, this technique does not always result in germline transgenic offspring and has a low success rate for livestock. Alternate approaches, such as somatic cell nuclear transfer using transgenic fibroblasts, do not show an increase in efficiency compared to PNI, while viral-based transgenesis is hampered by issues regarding transgene size and biosafety considerations. We have recently described highly successful transgenesis experiments with mice using a piggyBac transposase-based vector, pmhyGENIE-3. This construct, a single and self-inactivating plasmid, contains all the transpositional elements necessary for successful gene transfer. In this series of experiments, our laboratories have implemented cytoplasmic injection (CTI) of pmGENIE-3 for transgene delivery into in vivo-fertilized pig zygotes. More than 8.00% of the injected embryos developed into transgenic animals containing monogenic and often single transgenes in their genome. However, the CTI technique was unsuccessful during the injection of in vitro-fertilized pig zygotes. In summary, here we have described a method that is not only easy to implement, but also demonstrated the highest efficiency rate for nonviral livestock transgenesis.

Urokinase-type plasminogen activator is a serine protease that is clinically used in humans for the treatment of thrombolytic disorders and vascular diseases such as acute ischemic stroke and acute peripheral arterial occlusion. This study explored the feasibility of using chickens as a bioreactor for producing human urokinase-type plasminogen activator (huPA). Recombinant huPA gene, under the control of a ubiquitous Rous sarcoma virus promoter, was injected into the subgerminal cavity of freshly laid chicken eggs at stage X using the replication-defective Moloney murine leukemia virus (MoMLV)-based retrovirus vectors encapsidated with VSV-G (vesicular stomatitis virus G) glycoprotein. A total of 38 chicks, out of 573 virus-injected eggs, hatched and contained the huPA gene in their various body parts. The mRNA transcript of the huPA gene was present in various organs, including blood and egg, and was germ-line transmitted to the next generation. The level of active huPA protein was 16-fold higher in the blood of the transgenic chicken than in the nontransgenic chicken (P < 0.05). The expression of huPA protein in eggs increased from 7.82 IU/egg in the G0 generation to 17.02 IU/egg in the G1 generation. However, huPA-expressing embryos had reduced survival and hatchability at d 18 and 21 of incubation, respectively, and the blood clotting time was significantly higher in transgenic chickens than their nontransgenic counterparts (P < 0.05). Furthermore, adult transgenic rooster showed reduced (P < 0.05) fertility, as revealed by reduced volume of semen ejaculate, sperm concentration, and sperm viability. Taken together, our data suggest that huPA transgenic chickens could be successfully produced by the retroviral vector system. Transgenic chickens, expressing the huPA under the control of a ubiquitous promoter, may not only be used as a bioreactor for pharming of the huPA drug but also be useful for studying huPA-induced bleeding and other disorders.

Short-term storage of embryos at low temperature induces developmental arrest of the embryo and would appear to be a valuable aid in embryo-transfer techniques to avoid wasting embryos. Embryo storage at 4°C was examined to allow synchronization with embryo-transfer recipients using the microinjection technique. Superovulation was induced in female Japanese White donor rabbits four days before mating with males. At the same time, control recipients were injected with human chorionic gonadotropin (hCG) to allow synchronization (R1); the hCG injections were delayed by 24 h in the experimental group (R2). DNA constructs for expressing human C-reactive protein or apolipoprotein AII were microinjected into the male pronuclei of the ova. The microinjected embryos were immediately transferred to recipients (R1) or stored at 4°C in phosphate-buffered saline containing 10% fetal bovine serum. After 17-20 h, the stored embryos were incubated at 37°C for one hour, and the morphologically normal embryos were transferred to recipients (R2). In the R1 rabbits, 855 embryos were transferred to 29 recipients, and 72.4% of the recipients became pregnant. Seven of the 84 offspring were transgenic. In the R2 rabbits, 478 embryos were transferred to 16 recipients, and 62.5% of the recipients became pregnant. Two of the 39 offspring were transgenic. There were no differences in pregnancy rate, litter size and transgenic integration rate between R1 and R2. These results suggest that the short-term 4°C storage of microinjected embryos can be a valuable method for synchronization with recipients, and reducing wastage of embryos and the sacrifice of rabbits.

The objective of this study was to examine the effect of donor breed on pronuclear-stage embryo yield to be used for DNA microinjection in a transgenesis goat program. Twelve Canindé and twelve Saanen goats were heat synchronized using a progestagen-cloprostenol treatment. Forty-eight hours before the sponge removal, superovulation was induced with a total administration of 4.4 mg/kg bodyweight NIH-FSH-P1, given twice daily in decreasing doses over 3 days. In addition, goats received 100 μg of GnRH and they were hand-mated at 36 and 48 h after progestagen removal. Embryo recovery was performed by oviduct flushing at 72 h after sponge removal. Embryos were microinjected with a DNA construct and noticeable swelling of the nuclei was the criterion for successful microinjection. The total diameter, cytoplasm diameter, zona pellucida thickness and pronuclei diameter were measured for each microinjected embryo. A higher (p < 0.05) percentage of fertilized ova was observed in Canindé (89.9%) than Saanen (36.2%) goats. In addition, Canindé donors produced a higher percentage of pronuclear embryos when compared with Saanen: 72.5% vs 20.6% (p < 0.05), respectively. Successful microinjection was verified in 96.7% and 73.3% of times in Canindé and Saanen embryos, respectively (p < 0.05). Significant differences were observed for all morphometric parameters except for cytoplasm diameter. In conclusion, under our study experimental conditions, Canindé were more efficient than Saanen goats concerning the pronuclear embryo yield and manipulation. The use of Canindé goats in transgenesis programs could be increase the interest in their breeding and could be contribute to saving them from extinction., (© 2009 Blackwell Verlag GmbH.)

Purpose: Quantitative in vivo imaging of lung perfusion in rodents can provide critical information for preclinical studies. However, the combined challenges of high temporal and spatial resolution have made routine quantitative perfusion imaging difficult in small animals. The purpose of this work is to demonstrate 4D micro-CT for perfusion imaging in rodents at heartbeat temporal resolution and isotropic spatial resolution., Methods: We have recently developed a dual tube/detector micro-CT scanner that is well suited to capture first pass kinetics of a bolus of contrast agent used to compute perfusion information. Our approach is based on the paradigm that similar time density curves can be reproduced in a number of consecutive, small volume injections of iodinated contrast agent at a series of different angles. This reproducibility is ensured by the high-level integration of the imaging components of our system with a microinjector, a mechanical ventilator, and monitoring applications. Sampling is controlled through a biological pulse sequence implemented in LABVIEW. Image reconstruction is based on a simultaneous algebraic reconstruction technique implemented on a graphic processor unit. The capabilities of 4D micro-CT imaging are demonstrated in studies on lung perfusion in rats., Results: We report 4D micro-CT imaging in the rat lung with a heartbeat temporal resolution (approximately 150 ms) and isotropic 3D reconstruction with a voxel size of 88 microm based on sampling using 16 injections of 50 microL each. The total volume of contrast agent injected during the experiments (0.8 mL) was less than 10% of the total blood volume in a rat. This volume was not injected in a single bolus, but in multiple injections separated by at least 2 min interval to allow for clearance and adaptation. We assessed the reproducibility of the time density curves with multiple injections and found that these are very similar. The average time density curves for the first eight and last eight injections are slightly different, i.e., for the last eight injections, both the maximum of the average time density curves and its area under the curve are decreased by 3.8% and 7.2%, respectively, relative to the average time density curves based on the first eight injections. The radiation dose associated with our 4D micro-CT imaging is 0.16 Gy and is therefore in the range of a typical micro-CT dose., Conclusions: 4D micro-CT-based perfusion imaging demonstrated here has immediate application in a wide range of preclinical studies such as tumor perfusion, angiogenesis, and renal function. Although our imaging system is in many ways unique, we believe that our approach based on the multiple injection paradigm can be used with the newly developed flat-panel slip-ring-based micro-CT to increase their temporal resolution in dynamic perfusion studies.

The unique accessibility of chicken primordial germ cells (PGCs) during early development provides the opportunity to combine the reproduction of live animals with genetic conservation. Male and female Gifujidori fowl (GJ) PGCs were collected from the blood of early embryos, and cryopreserved in liquid nitrogen for >6 months until transfer. Manipulated GJ embryos were cultured until hatching; fertility tests indicated that they had normal reproductive abilities. Embryos from two lines of White Leghorn (24HS, ST) were used as recipients for chimera production following blood removal. The concentration of PGCs in the early embryonic blood of 24HS was significantly higher than in ST (P < 0.05). Frozen-thawed GJ PGCs were microinjected into the bloodstream of same-sex recipients. Offspring originating from GJ PGCs in ST recipients were obtained with a higher efficiency than those originating from GJ PGCs in 24HS recipients (23.3% v. 3.1%). Additionally, GJ progeny were successfully regenerated by crossing germline chimeras of the ST group. In conclusion, the cryogenic preservation of PGCs from early chicken embryos was combined with the conservation of live animals.

This study was performed to produce transgenic Korean native goat (Capra hircus) by laparoscopic embryo transfer (ET) to overcome the limitations of ET performed by laparotomy. Transgenic embryos were produced by DNA pronuclear microinjection of in vivo zygotes. The recipient goats were synchronized for estrus by using an introvaginal progesterone devices as a controlled internal drug-releasing insert (CIDR) for 13 days and injection of 400 IU PMSG 48 h before removal of the insert. Embryos were transferred on day 3 and 4 after removal of the insert. Recipient goats were deprived of feed for 48 h, then suspended in a laparotomy cradle at an angle of 45 degrees . After obtaining a sufficient pneumoperitoneum, the laparoscope and forceps were inserted abdominally through 5 mm trocar sleeves. Examination of the ovaries and uterus was performed and then 213 embryos were transferred into the oviducts via the infundibula of 76 recipient goats. To compare pregnancy rates, ET was also performed by laparotomy in 82 recipient goats. The pregnancies in the recipient goats were diagnosed by ultrasound on day 30 after embryo transfer. The pregnancy rate with laparoscopic ET was significantly higher than with ET performed by laparotomy (46.1% vs. 28.6%, p < 0.05). In addition, the pregnancy rates were compared between ovulated and non-ovulated ovaries of the recipient goats in the laparoscopic ET group. No significant difference was observed between the pregnancy rates of ovulated and non-ovulated ovaries (41.3% vs. 33.3%, p < 0.05) suggesting that ET may also be possible in non-ovulated recipients through artificial rupture of Graafian follicles. These results suggest that laparoscopic ET is a highly efficient method for the transfer of goat embryos.

Superovulation is a fundamental procedure for generating transgenic rodents. While various methods exist, zygote yield/quality remain suboptimal, making these techniques open to refinement. All require a follicle stimulating and a luteinising effect. The former can be induced by pregnant mare serum gonadotrophin (PMSG) or other compounds like human menopausal gonadotrophin (HMG). While HMG can double zygote yield compared to PMSG, no study has compared their effects on embryo quality. Embryo yield could also be increased with PMSG: timing administration at estrus may further improve follicular recruitment. This study compared: (i) the efficacy of HMG/PMSG for producing viable embryos for microinjection; and (ii) the effect of HMG/PMSG administration at estrus on embryo yield. Whitten effect-induced estrous C57/Bl6xCBA F(1) hybrid mice were superovulated as follows: PMSG (day 1; 5 IU intraperitoneally) or HMG (days 1 and 2; 1 IU intramuscularly); all received human chorionic gonadotrophin (hCG) on day 3 (5 IU, intraperitoneally). Zygotes were retrieved following mating, morphologically assessed and microinjected with innocuous ZhAT1R construct (expressing LacZ reporter and human angiotensin II type 1 receptor) before transfer to pseudopregnant recipients. Pups were tested for the transgene by Southern blot. Neither HMG nor PMSG proved superior in improving embryo yield, morphology and short-term post-microinjection survival. However, HMG group micromanipulated embryos all failed to establish a pregnancy/generate transgenic pups, unlike their PMSG counterparts. While HMG can be used for superovulation, it appears to increase embryo vulnerability to the long-term effects of microinjection. Furthermore, the embryo yields associated with HMG can be replicated by timing PMSG injection to coincide with Whitten effect-induced estrus.

Studies in rodents show that spermatogonial transplantation is an excellent new tool for studying spermatogenesis and for preservation and dissemination of genetics. The aim of this study was to adapt the technique to rams. Two issues were addressed: purification of stem cell spermatogonia, and efficient injection of donor spermatogonia into the seminiferous tubules of rams. We compared differential plating and Percoll gradient methods for purifying donor spermatogonia from ram lamb testes. Spermatogonia were identified with an antibody against PGP 9.5, a ubiquitin C-terminal hydrolase. Both purity and total number of spermatogonia recovered were higher after purification by Percoll gradient than by differential plating. Four approaches for injecting cells into the seminiferous tubules of ram testes were compared ex vivo: insertion of a needle into the extra-testicular rete testis after reflection of the head of the epididymis ('surgical' approach), and ultrasound-guided insertion of a needle into the extra-testicular rete, and the proximal and distal parts of the intra-testicular rete testis. 'Surgical' and ultrasound-guided approaches into the extra-testicular rete resulted in highest success rates and best filling of the seminiferous tubules. Finally, the ultrasound guided approach into the extra-testicular rete testis was validated in vivo by transplanting purified spermatogonia previously labeled with a fluorescent molecule (CFDA-SE). In seven of eight testes injected, donor cells were identified within the seminiferous epithelium for up to 2wk after transplantation, indicating the integration of donor cells.

The objective of this study was to explore the feasibility of employing adult fibroblasts as donor cells in interspecies nuclear transfer (NT) between buffaloes and cattle. Buffalo and bovine oocytes matured in vitro for 22 h were enucleated by micromanipulation using the Spindle View system. An ear fibroblast, pretreated with 0.1 microg/mL aphidicolin for 24 h, followed by culture for 2-9 days in Dulbecco's Modified Eagle's Media+0.5% fetal bovine serum, was introduced into the cytoplast by microinjection. Reconstructed oocytes were activated by exposure to 5 microM ionomycin for 5 min and 2 mM 6-dimethylaminopurine for 3 h. When buffalo adult fibroblasts were used as donor cells, there were no differences (P < 0.75) in the cleavage rate (66.2% versus 64.0%) between bovine and buffalo recipient oocytes, but more embryos derived from bovine cytoplasts developed to blastocysts than from buffalo cytoplasts (13.3% versus 3.0%, P < 0.05). When bovine adult fibroblasts were used as donor nuclei, both cleavage rate (45.3%) and blastocyst yield (4.5%) of NT embryos derived from buffalo cytoplasts were lower than those of NT embryos derived from bovine cytoplasts (65.5 and 11.9%, P < 0.05). The proportion of parthenogenetic buffalo (29.1%) or bovine (35.6%) oocytes developing to blastocysts was higher than those of NT embryos (P < 0.01). Interspecies NT embryos were derived from the donor cells and 55.0-61.9% of them possessed a normal diploid karyotype. In conclusion, embryos reconstructed by interspecies NT of adult fibroblasts between buffaloes and cattle developed to blastocysts, but bovine cytoplasts may direct embryonic development more effectively than buffalo cytoplasts, regardless of donor cell species.

The objective of the study reported here was to use scanning electron microscopy (SEM) to discover and describe the details of three-dimensional profiles and the natural (not surgically disturbed) topography/location of the infundibulum in the mouse. It will help new investigators to more quickly identify the infundibulum for successful transfer of microinjected eggs through a small opening into the oviduct/ampulla of pseudopregnant female mice for producing transgenic mice. Results of the study also illustrate the geographic orientation and natural topographic features of the ovary, infundibulum, ampulla, oviduct, and uterus. The presence of cilia on the surface of the crown foldings in the longitudinal section of the infundibular head stained with 1% toluidine blue provided direct evidence that evagination of the internal cilia of the infundibulum/oviduct results in formation of the infundibular crown. The new observation of the narrowing region of the infundibular head after surgical removal of the crown also suggests that formation of the infundibular crown may have resulted from the "evagination process" of internal cilia of the infundibulum/oviduct surface. The results also provide new evidence that the crown, terminal opening, and appearance of the left and the right infundibula of the same mouse differ.

The effects of sublethal exposure of a commercial blend of polychlorinated biphenyls (PCB), i.e. Clophen A50, on disease resistance to the aetiological agent of rainbow trout fry syndrome, Flavobacterium psychrophilum, were investigated. Newly fertilised rainbow trout Oncorhynchus mykiss eggs were nanoinjected with 2 doses of Clophen A50 (0.4 or 2 microg egg(-1)) and/or 100 colony forming units of F. psychrophilum. The mean cumulative mortality in control groups, and groups exposed to the lower dose of Clophen A50 (0.4 microg egg(-1)) was below 5.0%. The mean cumulative mortality in groups exposed to the higher dose of Clophen A50 (2.0 microg egg(-1)) was 5.8%, which was not significantly different from the control groups. In all groups infected with F. psychrophilum, with or without exposure to Clophen A50, significantly higher cumulative mortalities compared with control groups were recorded. No differences in mortality were recorded between groups exposed to bacteria alone or bacteria in combination with the higher dose of Clophen A50 (21.6 and 20.4%, respectively). Decreased disease resistance was recorded in groups exposed to F. psychrophilum and the lower dose of Clophen A50, with a mean cumulative mortality of 56.0%. These results could be due to non dose-dependent effects on the immune system, or toxic effects of PCB or their metabolites on the bacteria in groups exposed to the higher dose of Clophen A50. The present study indicates that maternal transfer of PCB might affect disease resistance to vertically transmitted F. psychrophilum.

The objective of this study was to examine the feasibility of identification and selection of cattle embryos based on green fluorescence (GFP-positive) in order to obtain calves carrying an integrated transgene. The construct used (pbLGTNF-EGFP) contained the human tumor necrosis factor alpha (hTNFalpha) gene fused to the bovine beta-lactoglobulin promoter (bLG) in plasmid vector pCX-EGFP. In four experiments, 76 zygotes were injected; eight of them developed to the morulae/blastocysts stage of which only five were GFP positive (one of them 100%, one-50%, three- 25%). All of the GFP positive embryos were transferred to recipients. Two calves were born: one after transfer of the 100% GFP positive embryo and the other after transfer of one of the 25% GFP positive embryos. Both animals were healthy with normal weight when compared to two control calves. The integration of pbLGTNF-EGFP in the host genome could not be detected in either of the calves, suggesting that GFP is an unreliable marker for preimplantation screening of embryos.

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.

The cryoloop is a technique where a thin nylon loop is used to suspend a film of cryoprotectant containing the oocytes and directly immersing them in liquid nitrogen. 508 bovine oocytes were collected, of these 351 were cryopreserved by slow freezing using standard straws or a new vitrification method using our self-constructed cryoloops and the remainder were controls. After thawing, the oocytes were inseminated by ICSI or standard IVF. The cryoloop vitrification method yielded a survival rate of 90.5% and the slow freezing technique a rate of 54.4% (p < 0.0001). When ICSI was performed, cryopreservation by the cryoloop vitrification method resulted in very similar cleavage rate to controls (16.0% vs. 17.3%) but slow freezing produced a slightly lower rate (9.4%). Cleavage rates after IVF in fresh oocytes was higher than the cryopreservation groups (49.5% vs. 15.4% and 25.8%), whereas after ICSI the rates were similar in all groups (17.3% vs. 9.4% and 16%). It is concluded that the new cryoloop vitrification technique followed by ICSI produce good embryo formation results and they could hold the future for effective oocyte cryopreservation.

This article summarizes recent progress in reproductive biotechnology in swine with special reference to in vitro production of embryos, generation of identical multiples, and transgenic pigs useful for xenotransplantation. In vitro production (in vitro maturation, in vitro fertilization, and in vitro culture) of viable porcine embryos is possible, although with much lower success rates than in cattle. The main problems are insufficient cytoplasmic maturation of porcine oocytes, a high proportion of polyspermic fertilization and a low proportion of blastocysts that, in addition, are characterized by a low number of cells, hampering their development in vivo upon transfer to recipients. Microsurgical bisection of morula and blastocyst stage embryos leads to a 2 to 3% monozygotic twinning rate of the transferred demiembryos, which is similar to that in rabbits and mice but considerably lower than in ruminants. It was found that with decreasing quality an increasing proportion of demi-embryos did not possess an inner cell mass. Porcine individual blastomeres derived from 4- and 8-cell embryos can be cultured in defined medium to the blastocyst stage. Leukemia inhibitory factor has been shown to be effective at defined embryonic stages and supports the formation of the inner cell mass in cultured isolated blastomeres in a concentration-dependent manner. For maintaining pregnancies with micromanipulated porcine embryos, it is not necessary to transfer extraordinarily high numbers of embryos. Porcine nuclear transfer is still struggling from the inefficiency of producing normally functioning blastocysts. Blastomeres, blastocyst-derived cells, fibroblasts and granulosa cells have been employed as donor cells in porcine nuclear transfer and have yielded blastocysts. Recently, the generation of the first piglets from somatic cell nuclear transfer has been achieved. DNA-microinjection into pronuclei of porcine zygotes has reliably resulted in the generation of transgenic pigs, which have special importance for the production of valuable pharmaceutical proteins in milk and xenotransplantation. It has been demonstrated that by expression of human complement regulatory proteins in transgenic pigs the hyperacute rejection response occurring after xenotransplantation can be overcome in a clinically relevant manner. Although biotechnological procedures in swine have recently undergone tremendous progress, the development is still lagging behind that in cattle and sheep. With regard to genetic engineering, considerable progress will originate from the possibility of employing homologous recombination in somatic cell lines and their subsequent use in nuclear transfer. In combination with the increasing knowledge in gene sequences this will allow in the foreseeable future widespread use in the pig industry either for agricultural or biomedical purposes.

The introduction of foreign DNA into the genome of livestock and its stable integration into the germ line has been a major technical advance in agriculture. Production of transgenic livestock provides a method to rapidly introduce "new" genes into cattle, swine, sheep and goats without crossbreeding. It is a more extreme methodology, but in essence, not really different from crossbreeding or genetic selection in its result. Several recent developments will profoundly impact the use of transgenic technology in livestock production. These developments are: 1) the ability to isolate and maintain in vitro embryonic stem (ES) cells from preimplantation embryos, embryonic germ (EG) and somatic cells from fetuses; and somatic cells from adults, and 2) the ability to use these embryonic and somatic cells as nuclei donors in nuclear transfer or "cloning" strategies. Cell based (ES, EG, and somatic cells) strategies have several distinct advantages for use in the production of transgenic livestock that cannot be attained using pronuclear injection of DNA. There are many potential applications of transgenic methodology to develop new and improved strains of livestock. Practical applications of transgenesis in livestock production include enhanced prolificacy and reproductive performance, increased feed utilization and growth rate, improved carcass composition, improved milk production and/or composition and increased disease resistance. Development of transgenic farm animals will allow more flexibility in direct genetic manipulation of livestock.

For parthenogenetic activation as a model system of nuclear transfer, microinjection and electroporation as activation treatments in bovine metaphase II oocytes were administered to each of three groups as follows: control group (treatments with Ca2+, Mg2+ -free PBS+100 micro M EGTA), IP3 group (control+25 micro M IP3) and IP3+ ryanodine group (control+25 micro M IP3+10 mM ryanodine). In experiments using microinjection, no significant differences were observed between any of the developmental stages of the electroporation experiment. For electroporation, cleavage rates were significantly higher in the IP3+ryanodine group than in the IP3 or control group (85.6% vs 73.7% or 67.6%, respectively). In the subsequent stages of embryonic development, such as morula and blastocyst formation, the IP3 and ryanodine group exhibited significantly higher rates of morula fomation than the IP3 or control groups (40.6% vs 24.2% or 16.7%, respectively). Similarly, the rate of blastocyst formation in the IP3+ryanodine group was significantly higher than the control group (16.3% vs 6.9%) but did not differ significantly from the IP3 group (16.3% vs 9.5%). In nuclear transfer, activation was performed at 30 hpm by microinjection and elecroporation with 25 micro M IP3+ 10 mM ryanodine followed by 6-DMAP treatment. No significant differences were observed at any stage of embryonic development and none of the embryos activated by electroporation reached either the morula or blastocyst stage. However, 3.8% and 1.9% of embryos activated by microinjection sucessfully developed to the morula and blastocyst stages, respectively. In conclusion, activation treatments using IP3 and ryanodine are able to support the development of bovine parthenogenetic and reconstructed embryos.

Embryonic vascular patterns in zebrafish (Danio rerio) could be visualised by confocal microscopy coupled with microinjected fluorescent microbeads. This microangiographic technique was adopted here, for the first time, to study the effects of cadmium on cardiovascular development in zebrafish embryos. Zebrafish embryos were incubated in culture medium containing 100 microM cadmium from 5 h post fertilisation (hpf) to 48 hpf. At 48 hpf, embryos were examined for viability and occurrence of malformations. The 100 microM cadmium caused 32.21 +/- 3.65% mortality and 20.33 +/- 4.04% visible malformations in surviving embryos. In the remaining embryos with no visible signs of malformations, further assessments for less obvious abnormalities were performed. Assessments on craniofacial development were made by digital measurements on areas of brains and eyes. Cardiac development was assessed by immunostaining the heart with the antibody MF20 specific for myosin heavy chain. Body lengths of the embryos were also measured. Embryonic development of brains, eyes, hearts and body lengths of visibly healthy embryos in the cadmium treatment group showed no significant difference from the controls. Embryonic vasculature of these visibly healthy embryos was then studied by microinjecting fluorescent microbeads of diameter 0.02 microm into the circulation. All the cadmium treated embryos showed localised vascular defects in the dorsal aortae, segmental and cranial vessels while none of the control embryos showed any aberrant patterns in the networking of the vasculature. Improved image analyses on the anterior regions revealed that cadmium treated embryos had markedly less complex networks of cranial vessels with fewer vessels perfusing the craniofacial regions. The number of branch points in the vascular network was counted. In untreated embryos, there were 135.6 +/- 51 branches in the vasculature in entire body. In the cadmium treated embryos, there were 64.5+/-31 branches. The difference was significant when assessed with Student's t-test. It appeared that although cadmium did not cause any signs of external malformations in these visibly healthy embryos, nonetheless induced impaired branching and anastomsis of the cranial vessels. This study revealed, for the first time, that vital vascular structures in fish embryos could be affected by exposure to cadmium. This technique allowed visualisation of vascular anomalies in embryos showing no external signs of malformations. The impairment of anatomical features during embryonic development might serve as meaningful health endpoints in ecotoxicological studies and in risk assessment.

The effect of timing of microinjection of DNA constructs on the efficiency of transgenic embryo production and improved efficiency and quality through combining EGFP as a reporter gene with nuclear transfer techniques were examined. From 12 to 24 h after insemination, constructs of pCXNeo-EGFP were microinjected into a pronucleus of bovine IVM-IVF zygotes. Due to the difficulty in visualizing pronuclei, the incidence of successful injection of linear DNA was higher when zygotes were injected between 20 and 24 h, as compared with an early period between 12 and 16 h after insemination. However, developmental competence of DNA-injected zygotes and the EGFP expression rate were not affected by the injection time. A majority of the embryos expressing EGFP signal were mosaic. Following nuclear transfer of blastomeres expressing EGFP, 4.5% of morulae that developed from the NT eggs had a strong EGFP signal in all live blastomeres. In other embryos, EGFP signal had been lost. When cells derived from the EGFP-positive NT morulae were subcultured, all the cells expressed strong EGFP signal at the second passage and demonstrated neomycin resistance. These results show that transient expression of nonintegrated EGFP appears frequently in EGFP-positive bovine embryos and that additional selection of EGFP-positive morulae after nuclear transfer of EGFP-positive blastomeres would facilitate selection of transgenic embryos.

Superovulation of female rabbits was induced by subcutaneous injection(s) of porcine FSH. Zygotes were recovered 17 to 19 hr after hCG injection and were classified into two categories under a microscope equipped with Nomarski interference-contrast optics at x 200 magnification: (A) zygotes with clearly visible pronuclei, or (B) zygotes with visualized pronuclei after 10 min centrifugation at 12,000 x g. No significant difference between strains was found in the proportion of category-A zygotes (JW 72.6% vs NZW 79.3%). Pronuclei of category-A zygotes were located in the center of the cytoplasm, and the pronuclei of category-B zygotes were slightly moved by centrifugation toward the mass of cytoplasmic lipid droplets. Exogenous DNA solution (5 microg/ml of fusion gene composed of bovine alphaS1-casein promoter and human growth hormone structural gene) was microinjected into the pronucleus of the JW zygotes. The pronucleus of category-A zygotes with a mean volume of 7.4 pl swelled up to 16.6 pl (132% increase), while that of category-B zygotes with a mean volume of 6.1 pl swelled up to 15.9 pl (148% increase). Nevertheless, similar proportions of category-A and category-B zygotes developed into offspring after transfer to recipient females (11.1 and 11.2%, respectively). The efficiency to produce hGH-carrying transgenic rabbits was 0.9% (2/235) from category-A zygotes and 0.5% (1/215) from category-B zygotes (P>0.05). To date, transgenic rabbits have been produced without centrifugation of pronuclear zygotes. However approximately 25% of fertilized rabbit zygotes can be used for DNA microinjection after they have been centrifuged to visualize their pronuclei.

In our continuing quest to improve the efficiency of producing transgenic animals, we have compared the influence of two transgene purification techniques on the efficiency of creating transgenic sheep and mice. Three hundred eighty-seven sheep zygotes and 2,737 mouse zygotes were microinjected with one of four transgenes. Transgenes were isolated from plasmid sequences either by agarose gel electrophoresis followed by gel extraction or by a single step sodium chloride gradient fractionation technique. Four transgenic sheep and 61 transgenic mice were produced. Both sheep and mice embryos responded similarly to transgene preparation methods. Overall, pregnancy rate was higher for recipients that received embryos injected with NaCl purified DNA (mean +/- SEM: 64 +/- 7% vs. 38 +/- 7%). Furthermore, offspring per zygote transferred (NaCl, 22 +/- 3% vs. Gel, 12 +/- 3%) and transgenics born per zygote transferred (NaCl, 3.9 +/- 0.6% vs. Gel, 1.5 +/- 0.6%) were higher when the NaCl purified DNA was used. However, the proportion of offspring born that were identified as transgenic did not differ between transgene purification methods. Transgenes responded differently to methods of preparation. One of the four genes yielded a significantly higher proportion of transgenics when the transgene was prepared by NaCl purification. These data suggest that on average the NaCl gradient purification technique results in a higher embryo survival rate to term for both sheep and mice, but the technique has no influence on rate of transgene integration.

Nuclear transfer as originally developed for use in amphibians involved microinjecting a nucleus directly into the cytoplasm of the oocyte. A major mammalian modification has been to use cell fusion to introduce the nucleus. Here we report using a microinjection method to introduce small and medium sized fibroblast cells into mature oocytes. Small cells were more likely to result in nuclear formation (30%) than larger cells (15%; P = 0.013). Small, confluent and serum starved cells resulted in nuclear formation more often (P < 0.048) than did cycling cells. The rate of nuclear formation was not dependent upon the media, (NCSU-23 or TL-Hepes without calcium) nor upon the duration of exposure to the media (1 h to 4 h) after microinjection but before activation. While such treatments did not have an effect on nuclear formation, treatment of parthenogenetically activated oocytes with calcium-free TL-Hepes reduced the percentage of blastocysts (P = 0.068. 11.2% vs. 18.3%) and increased the percentage of morula stage embryos (P = 0.007; 27.6% vs. 15.7%) as compared with culture in NCSU. Finally, small confluent cells were used for nuclear transfer and resulted in two presumptive blastocyst stage embryos [2/128 injected or 2/38 (5.3%) successful injections]. These results show that presumptive blastocyst stage embryos can result from microinjection of fibroblast cells to enucleated oocytes and thus may provide a method to create transgenic knockout animals.

Transgenic technology permits major modifications of phenotype by introducing subtle changes in genotype. For domestic farm species, genetic modification may be used to enhance agricultural production or to generate novel genotypes capable of producing heterologous proteins for biomedical applications. The advent of in vitro embryo production techniques has facilitated the large-scale, commercial use of transgenic technology in cattle. Accordingly, we employed in vitro-produced zygotes and embryos in an effort to generate transgenic cattle. Overall, pronuclei in 36,530 in vitro matured and fertilized zygotes were microinjected with a construct designed to express human alpha-lactalbumin in the mammary gland. Of these, 1,472 developed and were transferred to recipients, including 148 twin transfers. Initial pregnancy rate on Day 30 of gestation was 28% (374/1,324). Subsequent calving rate was 17% (226/1,324). Eighteen calves (8%) were transgenic. In vitro produced embryos were used to facilitate breeding of transgenic bulls. Frequency of transgene transmission varied from 3 to 54% between bulls, indicating varying degrees mosaicism. Embryos produced in vitro by these bulls were biopsied and screened for transgenesis prior to transfer to recipients; so far all (6/6) calves born from screened, transgenic embryos were themselves transgenic.

More abnormal fertilization has been found in sheep oocytes after intracytoplasmic sperm injection (ICSI) than after in vitro fertilization (IVF). Although the birth of a normal lamb has been reported, the efficiency of blastocyst production is low. We therefore evaluated the cleavage, development and viability of sheep embryos obtained from ICSI, IVF and sham injection. In vitro matured oocytes either injected or inseminated with spermatozoa were assessed for cleavage 1 and 4 d after injection or insemination, and for development to blastocyst after 7 d of culture. A total of 699 oocytes was injected (ICSI); 198 (30.6%) were activated and 55 (8.5%) developed to the blastocyst stage. Of the 17 recipient ewes with 1, 2, 3 or 4 embryos, 15 (88.2%) were pregnant on Day 18; of these 17 recipients, 7 (41.1%) and 6 (35.2%) ewes remained pregnant on Days 45 and 110, respectively. Two normal lambs were born, one ewe died on Day 110 with 2 normal male fetuses, another ewe aborted on Day 90 and 4 pregnancies were maintained. A total of 517 oocytes was inseminated (IVF); 296 (62%) were activated and 90 (18.8%) reached the blastocyst stage. A total of 19 ewes received 1, 2, 3 or 4 embryos; of these, 13 (68.4%) were pregnant on Day 18, 8 (42.1%) ewes remained pregnant on each of Days 45 and 110. Three ewes delivered 5 lambs. Five pregnancies were maintained. A total of 156 oocytes was sham injected, 38 (24.3%) were activated and no blatocysts were obtained after culture. The results of this study showed that blastocysts obtained after ICSI are potentially viable and are not a result of parthenogenesis.

The technique of microinjection is so far the only successful method of gene transfer in farm animals. Fertilized oocytes are collected from superovulated donors by flushing the oviducts and the exogenous gene construct is injected into the male pronucleus of zygotes before transfer into the oviducts or uteri of synchronized recipients. The resulting pups have to be tested for integration and expression of the foreign gene. Potential applications of gene transfer in farm animals are the improvement of growth parameters, the increase of disease resistance, the enhancement of reproduction rates, the increase of wool production of sheep, the alteration of metabolic pathways, the production of human hemoglobin in the blood of transgenic animals, the generation of transgenic animals as organ donors and the proteins in the milk of transgenic animals. Many studies showed, that farm animals are able to direct the expression of partially large amounts of foreign proteins for biomedical purposes into their milk. Therefore transgenic livestock could be an attractive alternative for conventional production methods.

A series of experiments evaluated development of porcine zygotes microinjected with DNA in three culture media and two incubation temperatures, from postpubertal and prepubertal donors, and between zygotes injected with DNA into the pronucleus and the cytoplasm. Zygotes recovered from 36 postpubertal gilts in Exp. 1 were injected and cultured in modified NCSU-23, modified NCSU-37, and CZB media at 37 degrees C or 39 degrees C for 7 d. In Exp. 2, zygotes were collected from postpubertal or prepubertal gilts, microinjected with DNA, and cultured in modified NCSU-23. In Exp. 3 superovulated prepubertal gilts had DNA injected into the cytoplasm or pronucleus of zygotes. Mean percentages developing to the expanded or hatched blastocyst stage in modified NCSU-23 (42.9) and modified NCSU-37 (40.1) did not differ, but development was greater than that for zygotes cultured in CZB (8.8; P < .05). Development was greater at 39 degrees C (P < .05) than at 37 degrees C (36.5 vs 24.6%). Microinjection of DNA decreased development (P < .05) from that of noninjected controls (18.1 vs 43.1%). Zygotes from postpubertal gilts had a higher percentage (68.0) of expanded and hatched blastocysts than zygotes from prepubertal donors (29.0; P < .05). No development difference was found between DNA injection into the pronucleus (23.1%) or cytoplasm (17.4%), but development was less than for control embryos (64.9%; P < .05). DNA microinjected porcine zygotes can be successfully cultured to the expanded blastocyst stage in modified NCSU-23 and modified NCSU-37 media at 39 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

The ability to manipulate the genome of the whole animal has, for the past 10 years, provided researchers with an alternative route of inquiry into many complex biological processes. Transgenic animals have numerous applications, encompassing a wide range of different disciplines, but they have proved especially useful in the investigation of gene regulation and gene function within the context of the living animal. This review describes the different techniques which have been used to produce transgenic animals and highlights advances which have been achieved using the transgenic approach.

Bovine zygotes derived from oocytes matured and fertilized in vitro were cultured in vitro. In Exp. 1, zygotes were vortexed to remove cumulus cells and then cultured in oviduct epithelial cell-conditioned TCM-199 medium with or without added cumulus cells. Embryos cultured without cumulus cells developed to blastocysts significantly less often than those cultured with added cumulus cells, 3 vs 38% of cleaved ova (P < .05). Co-culture of embryos with cumulus cells during the first 48 h only, or for the duration of the culture period, resulted in development rates similar to those of controls that were not vortexed. In Exp. 2, denuded zygotes were centrifuged (15,850 x g, 3 min) at room temperature to facilitate visualization of pronuclei. Centrifuged zygotes were either placed directly into conditioned medium for culture (with or without added cumulus cells) or DNA was first microinjected into their cytoplasm or a pronucleus. All microinjected embryos were co-cultured with added cumulus cells. Centrifugation and microinjection did not reduce viability significantly when embryos were co-cultured with cumulus cells. Development was reduced significantly when embryos were cultured without cumulus cells. Thus, when oviduct epithelial cell-conditioned medium was used, cumulus cell co-culture was beneficial during the early cleavage stages of in vitro-derived zygotes, including those that received DNA microinjection.

Pronuclear microinjection was used to produce transgenic mice harboring gene constructs, in which 110 base pairs (WAP1) or 2.4 kilobases (WAP2) of the 5' flanking sequences of the whey acidic protein (WAP) gene were fused to human growth hormone (hGH)-coding sequences. Female WAP-hGH transgenic mice expressed the transgenes in the mammary gland, the expression of the WAP2-hGH transgene mirroring that of the endogenous WAP gene. When other organs were examined, high level expression of hGH was observed in the brains of WAP2-hGH transgenic mice. Using in situ hybridization and immunohistochemistry, hGH expression from the transgene was seen to occur specifically in Bergmann glia cells. While normally neither WAP nor hGH is expressed in this type of cell, it appears that a combination of the regulatory region of the WAP gene and the hGH structural gene results in a novel tissue specificity in Bergmann glia cells.