Traceability of primordial germ cells in three neotropical fish species aiming genetic conservation actions.

Primordial germ cells (PGCs) are embryonic pluripotent cells that can differentiate into spermatogonia and oogonia, and therefore, PGCs are a genetic source for germplasm conservation through cryobanking and the generation of germline chimeras. The knowledge of PGC migration routes is essential for transplantation studies. In this work, the mRNA synthesized from the ddx4 3'UTR sequence of Pseudopimelodus mangurus, in fusion with gfp or dsred, was microinjected into zygotes of three neotropical species (P. mangurus, Astyanax altiparanae, and Prochilodus lineatus) for PGC labeling. Visualization of labeled PGCs was achieved by fluorescence microscopy during embryonic development. In addition, ddx4 and dnd1 expressions were evaluated during embryonic development, larvae, and adult tissues of P. mangurus, to validate their use as a PGC marker. As a result, the effective identification of presumptive PGCs was obtained. DsRed-positive PGC of P. mangurus was observed in the hatching stage, GFP-positive PGC of A. altiparanae in the gastrula stage, and GFP-positive PGCs from P. lineatus were identified at the segmentation stage, with representative labeling percentages of 29% and 16% in A. altiparanae and P. lineatus, respectively. The expression of ddx4 and dnd1 of P. mangurus confirmed the specificity of these genes in germ cells. These results point to the functionality of the P. mangurus ddx4 3'UTR sequence as a PGC marker, demonstrating that PGC labeling was more efficient in A. altiparanae and P. lineatus. The procedures used to identify PGCs in P. mangurus consolidate the first step for generating germinal chimeras as a conservation action of P. mangurus.

Additive Manufactured Piezoelectric-Driven Miniature Gripper.

In several cases, it is desirable to have prototypes of low-cost fabrication and adequate performance. In academic laboratories and industries, miniature and microgrippers can be very useful for observations and the analysis of small objects. Piezoelectrically actuated microgrippers, commonly fabricated with aluminum, and with micrometer stroke or displacement, have been considered as Microelectromechanical Systems (MEMS). Recently, additive manufacture using several polymers has also been used for the fabrication of miniature grippers. This work focuses on the design of a piezoelectric-driven miniature gripper, additive manufactured with polylactic acid (PLA), which was modeled using a pseudo rigid body model (PRBM). It was also numerically and experimentally characterized with an acceptable level of approximation. The piezoelectric stack is composed of widely available buzzers. The aperture between the jaws allows it to hold objects with diameters lower than 500 µm, and weights lower than 1.4 g, such as the strands of some plants, salt grains, metal wires, etc. The novelty of this work is given by the miniature gripper's simple design, as well as the low-cost of the materials and the fabrication process used. In addition, the initial aperture of the jaws can be adjusted, by adhering the metal tips in the required position.

Horse Somatic Cell Nuclear Transfer Using Zona Pellucida-Enclosed and Zona-Free Oocytes.

Horse cloning by somatic cell nuclear transfer (SCNT) is an attractive scientific and commercial endeavor. Moreover, SCNT allows generating genetically identical animals from elite, aged, castrated, or deceased equine donors. Several variations in the horse SCNT method have been described, which may be useful for specific applications. This chapter describes a detailed protocol for horse cloning, thus including SCNT protocols using zona pellucida (ZP)-enclosed or ZP-free oocytes for enucleation. These SCNT protocols are under routine use for commercial equine cloning.

Genome transfer technique for bovine embryo production using the metaphase plate and polar body.

Despite many studies in humans and mice using genome transfer (GT), there are few reports using this technique in oocytes of wild or domestic animals. Therefore, we aimed to establish a GT technique in bovine oocytes using the metaphase plate (MP) and polar body (PB) as the sources of genetic material. In the first experiment, GT was established using MP (GT-MP), and a sperm concentration of 1 × 106 or 0.5 × 106 spermatozoa/ml gave similar fertilization rates. The cleavage rate (50%) and blastocyst rate (13.6%) in the GT-MP group was lower than that of the in vitro production control group (80.2% and 32.6%, respectively). The second experiment evaluated the same parameters using PB instead of MP; the GT-PB group had lower fertilization (82.3% vs. 96.2%) and blastocyst (7.7% vs. 36.8%) rates than the control group. No differences in the amount of mitochondrial DNA (mtDNA) were observed between groups. Finally, GT-MP was performed using vitrified oocytes (GT-MPV) as a source of genetic material. The cleavage rate of the GT-MPV group (68.4%) was similar to that of the vitrified oocytes (VIT) control group (70.0%) and to that of the control IVP group (81.25%, P < 0.05). The blastocyst rate of GT-MPV (15.7) did not differ neither from the VIT control group (5.0%) nor from the IVP control group (35.7%). The results suggested that the structures reconstructed by the GT-MPV and GT-PB technique develop in embryos even if vitrified oocytes are used.

Establishing a model fish for the Neotropical region: The case of the yellowtail tetra Astyanax altiparanae in advanced biotechnology.

The use of model organisms is important for basic and applied sciences. Several laboratory species of fishes are used to develop advanced technologies, such as the zebrafish (Danio rerio), the medaka (Oryzias latipes), and loach species (Misgurnus spp.). However, the application of these exotic species in the Neotropical region is limited due to differences in environmental conditions and phylogenetic distances. This situation emphasizes the establishment of a model organism specifically for the Neotropical region with the development of techniques that may be applicable to other Neotropical fish species. In this work, the previous research efforts are described in order to establish the yellowtail tetra Astyanax altiparanae as a model laboratory species for both laboratory and aquaculture purposes. Over the last decade, starting with artificial fertilization, the yellowtail tetra has become a laboratory organism for advanced biotechnology, such as germ cell transplantation, chromosome set manipulation, and other technologies, with applications in aquaculture and conservation of genetic resources. Nowadays, the yellowtail tetra is considered the most advanced fish with respect to fish biotechnology within the Neotropical region. The techniques developed for this species are being used in other related species, especially within the characins class.

Embryo manipulation in neotropical characiform fish: incubation system, anaesthetic, and PGC transplantation in Prochilodus lineatus.

Primordial germ cells transplantation is a unique approach for conservation and reconstitution of endangered fish species. This study aimed to establish techniques to culture dechorionated embryos in different incubation systems and also to determine anaesthetic concentration for fish recipients in the larval stage for subsequent primordial germ cell transplantation. Intact and dechorionated embryos were divided into three incubation systems: (1) a control group with manual replacement of the solution; (2) a closed environment with high oxygen with manual replacement of the solution; and (3) constant solution recirculation. This combination resulted in six treatments. For the evaluation of anaesthetics for larvae, the concentrations evaluated were 19.5 mM, 24.4 mM, 29.3 mM, and 34.2 mM of 2-phenoxyethanol. Anaesthesia concentration and recovery at different stages were evaluated. For transplantation, primordial germ cells of Astyanax altiparanae were transplanted into anaesthetised larvae (1 dph) of Prochilodus lineatus. Better results were obtained in the recirculation system for dechorionated embryos of P. lineatus for hatching (54.18%) and normal morphology (50.06%). The 2-phenoxyethanol anaesthetic with a dose of 29.3 mM resulted in shorter induction times, in addition to the recovery time between 5 and 10 min. By using this anaesthetic concentration at transplantation, GFP-positive cells were seen in two recipients, but the cells did not proliferate. This study established an effective incubation system for the development of the dechorionated embryo and determined an effective anaesthetic concentration for P. lineatus larvae. In addition, micromanipulation and transplantation of primordial germ cells in neotropical species were conducted for the first time.

Primordial germ cell identification and traceability during the initial development of the Siluriformes fish Pseudopimelodus mangurus.

Primordial germ cells (PGCs) are responsible for generating all germ cells. Therefore, they are essential targets to be used as a tool for the production of germline chimeras. The labeling and route of PGCs were evaluated during the initial embryonic development of Pseudopimelodus mangurus, using whole-mount in situ hybridization (WISH) and mRNA microinjection in zygotes. A specific antisense RNA probe constituted by a partial coding region from P. mangurus nanos3 mRNA was synthesized for the WISH method. RNA microinjection was performed using the GFP gene reporter regulated by translation regulatory P. mangurus buc and nanos3 3'UTR sequences, germline-specific markers used to describe in vivo migration of PGCs. Nanos3 and buc gene expression was evaluated in tissues for male and female adults and initial development phases and larvae from the first to seventh days post-hatching. The results from the WISH technique indicated the origin of PGCs in P. mangurus from the aggregations of nanos3 mRNA in the cleavage grooves and the signals obtained from nanos3 probes corresponded topographically to the migratory patterns of the PGCs reported for other fish species. Diffuse signals were observed in all blastomeres until the 16-cell stage, which could be related to the two sequences of the nanos3 3'UTR observed in the P. mangurus unfertilized egg transcriptome. Microinjection was not successful using GFP-Dr-nanos1 3'UTR mRNA and GFP-Pm-buc 3'UTR mRNA and allowed the identification of potential PGCs with less than 2% efficiency only and after hatching using GFP-Pm-nanos3 3'UTR. Nanos3 and buc gene expression was reported in the female gonads and from fertilized eggs until the blastula phase. These results provide information about the PGC migration of P. mangurus and the possible use of PGCs for the future generation of germline chimeras to be applied in the conservation efforts of Neotropical Siluriformes species. This study can contribute to establishing genetic banks, manipulating organisms, and assisting in biotechnologies such as transplanting germ cells in fish.

Manual collapse of blastocoels in not effective in increasing the viability of vitrified equine embryos / Colabamento manual da blastocele não foi efetivo para aumentar a viabilidade de embriões equinos vitrificados

Embryo cryopreservation methods have been used for commercialization and formation of genetic banks. Cryopreservation of equine embryos <300 µm in diameter, collected at days 6-6.5 after ovulation, allows satisfactory pregnancy rates. However, higher embryo collection rates in mares are obtained when uterine flush is performed between days 7 and 8 after ovulation when embryos are >300 µm in diameter, needing blastocoel collapse for satisfactory resistance to cryopreservation by vitrification. To evaluate the viability of simplified blastocoel collapse by embryo puncture with low technology and low-cost equipment, 22 embryos, collected at day 8 post-ovulation (D8), were allocated to the following groups: (1) micropuncture with a 30 G needle, assisted by a mechanical micromanipulator, before vitrification (n=4); (2) manual blade microsection before vitrification (n=6); (3) no manipulation prior to vitrification (n=8); and (4) freshly inovulated embryos (n=4). Despite the high re-expansion rates observed after vitrification, embryos manipulated prior to vitrification (groups MP and MS) did not result in pregnancy 25 days after transfer. On the other hand, embryos from groups NM (non-micromanipulated) and FR (freshly inovulated) resulted in pregnancies at 25 days. Under the conditions of the present study, manual blastocoel collapse was not efficient in increasing cryotolerance to vitrification among large embryos, requiring improvements to obtain pregnancies.(AU)

Métodos de criopreservação de embriões têm sido utilizados com diversos objetivos. Maiores taxas de coleta embrio-nária em éguas com lavagem uterina realizada 7 a 8 dias pós ovulação. A criopreservação de embriões equinos com diâmetro <300 µm (6-6,5 dias após a ovulação) permite a obtenção de taxas de prenhez satisfatórias. Embriões com diâmetro >300 µm (7º dia pós-ovulação) somente são adequadamente criopreservados quando submetidos a colabamento da blastocele. Objetivando avaliar a viabilidade da punção da blastocele com equipamento de baixa sofisticação e custo, 22 embriões coletados no 8º. dia pós-ovulação (D8) foram alocados aos seguintes grupos: (1) micropunção com uma agulha 30 G assistida por micromanipula-dor antes da vitrificação (n=4); (2) microssecção manual por lâmina antes da vitrificação (n=6); (3) sem manipulação anterior à vitrificação (n=8); e (4) transferidos a fresco (n=4). Apesar de altas taxas de reexpansão após a criopreservação, os embriões manipulados previamente a vitrificação não resultaram em prenhez aos 25 dias. Tanto os embriões não micromanipulados, quanto os transferidos a fresco resultaram em prenhezes aos 25 dias. A microssecção manual não se mostrou eficiente como método para aumento da criotolerância de embriões grandes, necessitando um aprimoramento visando a obtenção de prenhezes.(AU)

Plant Chromosome-Specific Probes by Microdissection of a Single Chromosome: Is That a Reality?

Painting plant chromosomes through chromosomal in situ suppression (CISS) hybridization has long been considered impracticable. Seeking to build specific and complex probes from a single microdissected chromosome, we employed human chromosomes as models to standardize all the necessary steps for application in plants. Human metaphases were used to define the adequate conditions for microdissection, chromosome DNA amplification and labeling through degenerate oligonucleotide-primed PCR, and in situ hybridization stringency. Subsequently, these methodologies were applied in the plant species Zea mays (chromosome 1) and Capsicum annuum (chromosome 7 or 8). The high quality of human and plant cytogenetic preparations and the meticulous standardization of each step, especially the most critical ones - microdissection and first round of DNA amplification - were crucial to eliminate the signs of non-specific hybridization and for direct application in plants. By overcoming these challenges, we obtained chromosome-specific probes, which allowed to achieve a clear and uniform painting of the entire target chromosomes with little or no background, evidencing their complexity and specificity. Despite the high amount of ubiquitous repetitive sequences in plant genomes, the main drawback for chromosome painting, we successfully employed our methodology on two plant species. Both have more than 80% repetitive sequences, which is compared to the human genome (66-69%). This is the first time that plant chromosome-specific probes were successfully obtained from a single A mitotic or meiotic microdissected chromosome. Thereby, we assume that chromosome painting through microdissection and CISS hybridization can now be considered a reality in the field of plant cytogenetics.

Protocol for chromosome-specific probe construction using PRINS, micromanipulation and DOP-PCR techniques

ABSTRACT Chromosome-specific probes have been widely used in molecular cytogenetics, being obtained with different methods. In this study, a reproducible protocol for construction of chromosome-specific probes is proposed which associates in situ amplification (PRINS), micromanipulation and degenerate oligonucleotide-primed PCR (DOP-PCR). Human lymphocyte cultures were used to obtain metaphases from male and female individuals. The chromosomes were amplified via PRINS, and subcentromeric fragments of the X chromosome were microdissected using microneedles coupled to a phase contrast microscope. The fragments were amplified by DOP-PCR and labeled with tetramethyl-rhodamine-5-dUTP. The probes were used in fluorescent in situ hybridization (FISH) procedure to highlight these specific regions in the metaphases. The results show one fluorescent red spot in male and two in female X chromosomes and interphase nuclei.

Transgenesis and gene edition in small ruminants / Transgênese e edição gênica em pequenos ruminantes

This review summarizes the main achievements with the use of transgenesis and genome editingtechnologies in sheep and goats. Transgenesis, also referred to as recombinant DNA (rDNA) technology, madepossible by the first time 30 years ago the addition of novel traits from a given species into a different one. Onthe other hand, more recently genome editing appears a much more precise method of making changes to thegenome of a plant, animal, or other living organism, allowing for the addition, substitution, or deletion ofspecific nucleotides in an organism’s genome. With transgenesis, the introduction of new DNA into anorganism’s genome was generally without control of the site of the genome in which the insertion of that rDNAconstruct would occur. With genome editing in contrast, researchers and developers of products can makespecific changes in precise locations of the genome. This concept was absolutely improved with the novelCRISPR/Cas system, making genome edition cheaper, more efficient, easier and affordable for every Laboratoryaround the world. This revolution that originally emerged from molecular biology and passed to biomedicine,has recently been applied to livestock and agriculture. In addition, the application of this technology in sheep,goats, pigs and cattle, also has been possible by the advance of assisted reproductive technologies for embryoproduction, micromanipulation, cryopreservation and transfer. In general, multidisciplinary approaches includingbasic research and technical improvements, participation of private actors and adequate regulation should bemerged to take advantage of this potent biotechnology in different countries.

Transgenesis and gene edition in small ruminants / Transgênese e edição gênica em pequenos ruminantes

This review summarizes the main achievements with the use of transgenesis and genome editingtechnologies in sheep and goats. Transgenesis, also referred to as recombinant DNA (rDNA) technology, madepossible by the first time 30 years ago the addition of novel traits from a given species into a different one. Onthe other hand, more recently genome editing appears a much more precise method of making changes to thegenome of a plant, animal, or other living organism, allowing for the addition, substitution, or deletion ofspecific nucleotides in an organisms genome. With transgenesis, the introduction of new DNA into anorganisms genome was generally without control of the site of the genome in which the insertion of that rDNAconstruct would occur. With genome editing in contrast, researchers and developers of products can makespecific changes in precise locations of the genome. This concept was absolutely improved with the novelCRISPR/Cas system, making genome edition cheaper, more efficient, easier and affordable for every Laboratoryaround the world. This revolution that originally emerged from molecular biology and passed to biomedicine,has recently been applied to livestock and agriculture. In addition, the application of this technology in sheep,goats, pigs and cattle, also has been possible by the advance of assisted reproductive technologies for embryoproduction, micromanipulation, cryopreservation and transfer. In general, multidisciplinary approaches includingbasic research and technical improvements, participation of private actors and adequate regulation should bemerged to take advantage of this potent biotechnology in different countries.(AU)

Evidence of heterozygosity and recombinant alleles in single cysts of Giardia duodenalis / Evidência de heterozigose e alelos recombinantes em cistos individualizados de Giardia duodenalis

Abstract Giardia duodenalis is divided into eight assemblages (named A to H). Isolates of assemblage A are divided into four sub-assemblages (AI, AII, AIII and AIV). While isolates of sub-assemblage AII are almost exclusively detected in human hosts, isolates of assemblage B are encountered in a multitude of animal hosts and humans. Here, we isolated single cysts of G. duodenalis from a human stool sample and found that one of them had overlaps of assemblage AII and B alleles and an unexpectedly high number of variants of the beta-giardin (Bg) and GLORF-C4 (OrfC4) alleles. In addition, one of the Bg alleles of that cyst had a fragment of sub-assemblage AII interspersed with fragments of assemblage B, thus indicating that this allele may be a recombinant between sequences A and B. Our results are unprecedented and put a check on the statement that different assemblages of G. duodenalis represent species with different host specificities.

Resumo A espécie Giardia duodenalis é dividida em oito grupos (nomeados de A a H). Isolados do grupo A são divididos em quatro subgrupos (AI, AII, AIII and AIV). Enquanto isolados do subgrupo AII são detectados quase exclusivamente em hospedeiros humanos, isolados do subgrupo B são encontrados em uma grande variedade de hospedeiros entre animais e humanos. Neste trabalho, foi constatado que, dentre diversos cistos individualizados de G. duodenalis provenientes de fezes de origem humana, um cisto continha os alelos AII e B e um número inesperado de variantes de alelos codificadores de beta giardina e GLORF-C4. Ainda, um dos alelos beta giardina desse cisto possuía fragmentos AII intercalando um fragmento B, indicando que esse alelo pode ser um recombinante entre alelos AII e B. Os resultados aqui apresentados são inéditos e colocam em dúvida o conceito atual de que os diferentes grupos de G. duodenalis representam espécies distintas com diferentes graus de especificidade por hospedeiros.

Evidence of heterozygosity and recombinant alleles in single cysts of Giardia duodenalis / Evidência de heterozigose e alelos recombinantes em cistos individualizados de Giardia duodenalis

Giardia duodenalis is divided into eight assemblages (named A to H). Isolates of assemblage A are divided into four sub-assemblages (AI, AII, AIII and AIV). While isolates of sub-assemblage AII are almost exclusively detected in human hosts, isolates of assemblage B are encountered in a multitude of animal hosts and humans. Here, we isolated single cysts of G. duodenalis from a human stool sample and found that one of them had overlaps of assemblage AII and B alleles and an unexpectedly high number of variants of the beta-giardin (Bg) and GLORF-C4 (OrfC4) alleles. In addition, one of the Bg alleles of that cyst had a fragment of sub-assemblage AII interspersed with fragments of assemblage B, thus indicating that this allele may be a recombinant between sequences A and B. Our results are unprecedented and put a check on the statement that different assemblages of G. duodenalis represent species with different host specificities.(AU)

A espécie Giardia duodenalis é dividida em oito grupos (nomeados de A a H). Isolados do grupo A são divididos em quatro subgrupos (AI, AII, AIII and AIV). Enquanto isolados do subgrupo AII são detectados quase exclusivamente em hospedeiros humanos, isolados do subgrupo B são encontrados em uma grande variedade de hospedeiros entre animais e humanos. Neste trabalho, foi constatado que, dentre diversos cistos individualizados de G. duodenalis provenientes de fezes de origem humana, um cisto continha os alelos AII e B e um número inesperado de variantes de alelos codificadores de beta giardina e GLORF-C4. Ainda, um dos alelos beta giardina desse cisto possuía fragmentos AII intercalando um fragmento B, indicando que esse alelo pode ser um recombinante entre alelos AII e B. Os resultados aqui apresentados são inéditos e colocam em dúvida o conceito atual de que os diferentes grupos de G. duodenalis representam espécies distintas com diferentes graus de especificidade por hospedeiros.(AU)

Multilocus amplification of genomic DNA from single cysts of Giardia duodenalis separated using micromanipulation technique.

Giardia duodenalis is divided into at least eight groups, named assemblages A to H. Assemblages A and B are the only ones able to infect humans and other mammals. The species status for these assemblies is a moot point, but has not gained general acceptance because sexual activity in Giardia is not completely understood. Heterozygosity in G. duodenalis can be detected through simultaneous identification of multiple loci in single cysts or trophozoites. In this paper, we describe a technique that enables simultaneous detection of fragments from four genes from single cysts of G. duodenalis recovered from stool samples. Each cyst from a fecal sample of human origin was separated, the DNA was extracted and amplified by means of multiplex PCR directed to four genes and the multiplex PCR product was further re-amplified using four single PCR (one for each gene). The following loci were detected: beta giardin (bg), GLORF-C4 (orfC4), triose phosphate isomerase (tpi) and glutamate dehydrogenase (gdh). This procedure should make it possible to investigate multiple genes from a single cyst of G. duodenalis assemblage A or B.

A rapid and reliable method for the clonal isolation of Acanthamoeba from environmental samples

Acanthamoeba are abundant in a wide range of environments, and some species are responsible for cutaneous infections, keratitis, and granulomatous amoebic encephalitis (GAE). The conventional detection and isolation of amoeba from clinical and environmental samples involves sampling and culture on non-nutrient Ágar medium. Although efficient, this system requires several transfers in order to eliminate contaminants, and is not appropriate for the isolation of individual amoeba from samples with a biodiverse community. In this study we propose an alternative method for the isolation of monocystic clones of Acanthamoeba. The propose method involves sampling, enrichment, encystment induction, and direct cysts micromanipulation and culture on Ágar plates.

Recent advances in micromanipulation and transgenesis in domestic mammals

Background: Intracytoplasmic sperm injection (ICSI) involves mechanical transfer of a single sperm cell into ooplasm. A new application has been recently found for ICSI, the production of transgenic animals. Since the birth of ''Dolly'', the first adult somatic cloned mammal, viable offspring has been produced by nuclear transfer in many species including cattle. The present review briefly summarizes our experience with ICSI and somatic cell nuclear transfer mainly to produce transgenic embryos, as well as for the generation of new micromanipulation technique. Review: We have evaluated different factors that affect SCNT and transgenesis including the chemical activator, the transfection event and the effect of recloning. Also, we included a brief description of the ICSI technique, which we used in five different species, examining its potential to produce transgenic embryos. Finally different strategies to produce transgenic animals were analyzed: ICSI- mediated gen transfer (ICSI-MGT), Injection of cumulus cell and ooplasmic vesicle incubated for 5 min with the transgene or injection of the plasmid alone. All of them were very efficient in exogenous DNA expression at embryo stages but resulted in mosaic embryos. We demonstrated that "ICSI-MGT" assisted by chemical activation is the only treatment of sperm mediated gen transfer capable to generated transgenic embryos in ovine. Besides, after ICSI-MGT, it is possible to obtain enhanced green fluorescent protein (EGFP)-expressing embryos in five diferent species: ovine, porcine, feline, bovine and equine. Our studies also established for the first time that short term transgene co-incubation with somatic cells can produce transgene-expressing mammalian SCNT embryos, and also that parthenogenic, eDNA- expressing embryos can be obtained by injection of vesicles or eDNA alone. Moreover, eDNA- -expressing embryos can be also obtained by cytoplasmic injection of vesicles in IVF zygotes, simplifying the traditional IVF pronuclear injection technique. We tried a further simplification of the technique in bovine oocytes and zygotes, by intracytoplasmically injecting them with eDNA-liposomes complexes. Approximately 70% of the cleaved embryos and 50% of the blastocysts expressed EGFP, when egfp­liposome was injected 16 h post-fertilization. Different approaches were assayed to reverse the mosaicism including a novel technique of gamete cloning. Our first approach consisted of the production of transgenic IVF embryos by vesicle microinjection to generate transgenic blastomeres to be used as donor cells for cloning. A high efficiency in mosaicism reversal and multiplication of transgenic embryos was attaineded. Other technique assayed was the separation of transgenic blastomeres followed by the aggregation of two-cell fused embryos or by the asynchronous younger blastomere successfully multiplied transgenic embryos, and theoretically reduces mosaicism rates in future offspring [15]. This technology can also be used to multiply embryos from animals with high genetic value. We demonstrated that a sperm and oocyte can be efficiently cloned. Green haploid androgenic blastomeres produced with the injection of a single sperm by egfp ICSI-MGT could be used to fertilized oocytes resulting in several homogeneous expressing embryos. This approach shows great potential because it allows for determination of the sex of the sperm nucleus prior to fertilization. It is also possible to clone previously transfected oocytes followed by the reconstruction of biparental bovine embryos to generate homogeneous transgene-expressing embryos. This review summarizes recent experiments in micromanipulation and gene transfer in domestic animals. The objective is not to exhaustedly describe the research done in this field but to present the promising methods recently developed or evaluated in our lab. Conclusion: Significant advancements have been made in the course of the recent years in micromanipulation and transgenesis techniques. In our lab we have been evaluating ICSI and Nuclear transfer mainly to produce transgenic embryos. We used also transgensis to apply or developed new micromanipulation technique in domestic animals linke sperm and oocyte cloning.