Bovine parthenogenotes produced by inhibition of first or second polar bodies emission

Parthenogenetic embryos are an ethically acceptable alternative for the derivation of human embryonic stem cells. In this work, we propose a new strategy to produce bovine parthenogenetic embryos inhibiting the emission of the first polar body during in vitro maturation, and allowing the extrusion of the second polar body during oocyte activation. Cytochalasin B, an inhibitor of actin microfilaments, was employed during in vitro maturation to inhibit first polar body emission or during parthenogenetic activation to block second polar body emission. Only one polar body was inhibited in each strategy in order to keep the diploid chromosome set. In experiment 1, the effect of cytochalasin B on in vitro maturation of bovine oocytes was evaluated. Most oocytes (77%) were arrested at a meiotic stage characterized by the presence of a large internal metaphase plate and absence of polar body. In experiment 2, development of embryos exposed to cytochalasin B during in vitro maturation (CytoB-IVM) or during activation (CytoB-ACT) was compared. Developmental rates did not differ between diploidization strategies, even when three agents were employed to induce activation. Both groups, CytoB-IVM and CytoB-ACT, tended to maintain diploidy. CytoB-IVM parthenogenesis could help to obtain embryos with a higher degree of homology to the oocyte donor.Fil: Bevacqua, Romina Jimena. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernández y Martín, Rafael. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Effect of collection-maturation interval time and pregnancy status of donor mares on oocyte developmental competence in horse cloning

The current limitations for obtaining ovaries from slaughterhouses and the low efficiency of in vivo follicular aspiration necessitate a complete understanding of the variables that affect oocyte developmental competence in the equine. For this reason, we assessed the effect on equine oocyte meiotic competence and the subsequent in vitro cloned embryo development of 1) the time interval between ovary collection and the onset of oocyte in vitro maturation (collection-maturation interval time) and 2) the pregnancy status of the donor mares. To define the collection-maturation interval time, collected oocytes were classified according to the slaughtering time and the pregnancy status of the mare. Maturation rate was recorded and some matured oocytes of each group were used to reconstruct zona free cloned embryos. Nuclear maturation rates were lower when the collection-maturation interval time exceeded 10 h as compared to 4 h (32/83 vs. 76/136, respectively; P = 0.0128) and when the donor mare was pregnant as compared to nonpregnant (53/146 vs. 177/329, respectively; P = 0.0004). Low rates of cleaved embryos were observed when the collection-maturation interval time exceeded 10 h as compared to 6 to 10 h (11/27 vs. 33/44, respectively; P = 0.0056), but the pregnancy status of donor mares did not affect cloned equine blastocyst development (3/49 vs. 1/27 for blastocyst rates of nonpregnant and pregnant groups, respectively; P = 1.00). These results indicate that, to apply assisted reproductive technologies in horses, oocytes should be harvested within approximately 10 h after ovary collection. Also, even though ovaries from pregnant mares are a potential source of oocytes, they should be processed at the end of the collection routine due to the lower collection and maturation rate in this group.Fil: Gambini, Andres. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; ArgentinaFil: Jarazo, Javier. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Karlanian, Florencia. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: de Stéfano, Adrian. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentin

Clones, quimeras y otros seres extraordinarios

En este artículo se describirán ejemplos de especies animales en las que se da la clonación o el quimerismo en forma natural. Se revisarán algunos de los trabajos pioneros de autores que inspiraron las líneas de investigación en las que he estado involucrado. Pero, primordialmente, se analizarán varios de los progresos que hemos alcanzado con diferentes equipos de colegas desde 1993. Se incluyen experimentos de fecundación in vitro (FIV), inyección intracitoplasmática de espermatozoides (ICSI), clonación, transgénesis y edición génica, realizadas en diferentes especies de mamíferos. La aplicación futura de estas tecnologías a la producción animal debería estar sujeta al seguimiento de pautas de bienestar animal y bioética, siendo fundamental nuestra capacidad de imaginación e innovación. Por otro lado, los animales que ya se han generado con algunos métodos (FIV, ICSI y clonación), cuyas figuras se presentan en esta revisión, son verdaderas postales del futuro que demuestran la factibilidad de las metodologías descriptas.Presentación del Dr. Bernardo J. Carrillo.Academia Nacional de Agronomía y Veterinari

Clones, quimeras y otros seres extraordinarios

En este artículo se describirán ejemplos de especies animales en las que se da la clonación o el quimerismo en forma natural. Se revisarán algunos de los trabajos pioneros de autores que inspiraron las líneas de investigación en las que he estado involucrado. Pero, primordialmente, se analizarán varios de los progresos que hemos alcanzado con diferentes equipos de colegas desde 1993. Se incluyen experimentos de fecundación in vitro (FIV), inyección intracitoplasmática de espermatozoides (ICSI), clonación, transgénesis y edición génica, realizadas en diferentes especies de mamíferos. La aplicación futura de estas tecnologías a la producción animal debería estar sujeta al seguimiento de pautas de bienestar animal y bioética, siendo fundamental nuestra capacidad de imaginación e innovación. Por otro lado, los animales que ya se han generado con algunos métodos (FIV, ICSI y clonación), cuyas figuras se presentan en esta revisión, son verdaderas postales del futuro que demuestran la factibilidad de las metodologías descriptas.Presentación del Dr. Bernardo J. Carrillo.Academia Nacional de Agronomía y Veterinari

Clones, quimeras y otros seres extraordinarios

En este artículo se describirán ejemplos de especies animales en las que se da la clonación o el quimerismo en forma natural. Se revisarán algunos de los trabajos pioneros de autores que inspiraron las líneas de investigación en las que he estado involucrado. Pero, primordialmente, se analizarán varios de los progresos que hemos alcanzado con diferentes equipos de colegas desde 1993. Se incluyen experimentos de fecundación in vitro (FIV), inyección intracitoplasmática de espermatozoides (ICSI), clonación, transgénesis y edición génica, realizadas en diferentes especies de mamíferos. La aplicación futura de estas tecnologías a la producción animal debería estar sujeta al seguimiento de pautas de bienestar animal y bioética, siendo fundamental nuestra capacidad de imaginación e innovación. Por otro lado, los animales que ya se han generado con algunos métodos (FIV, ICSI y clonación), cuyas figuras se presentan en esta revisión, son verdaderas postales del futuro que demuestran la factibilidad de las metodologías descriptas.Presentación del Dr. Bernardo J. Carrillo.Academia Nacional de Agronomía y Veterinari

Transgénesis: una moderna biotecnología reproductiva en animales de interés zootécnico

Los continuos avances en el conocimiento de la biología molecular han permitido un gran progreso de la ciencia, mediante la modificación genética tanto de virus, bacterias como de organismos superiores. Estos procedimientos de alta complejidad que comprenden a la ingeniería genética permiten identificar, reproducir, modificar y transferir material genético en células, tejidos u organismos. A su vez, las modernas técnicas reproductivas que han logrado incrementar progresivamente su eficiencia en los últimos años (inseminación artificial, sincronización hormonal de estros, fecundación "in vitro", fertilización asistida, transferencia de embriones, clonación, etc.), constituyen herramientas indispensables para seguir avanzando en las nuevas investigaciones referidas a la modificación genética en los animales. Se describen brevemente las diversas metodologías empleadas para la realización de transgénesis en animales de interés zootécnico.Continuous advances in the understanding of molecular biology have allowed great progress of science, through genetic modification of viruses, bacteria and higher organisms. These highly complex procedures that include genetic engineering enable to identify, reproduce, modify and transfer genetic material into cells, tissues or organisms. In turn, modern reproductive techniques that have managed to gradually increase their efficiency in recent years (artificial insemination, hormonal synchronization of estrus, fertilization "in vitro", assisted fertilization, embryo transfer, cloning, etc.) are essential tools for continue advancing in new research concerning genetic modification in animals. The various methodologies used to carry out transgenesis in animals of zootechnical interest are briefly described.Fil: Gibbons, A.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche; ArgentinaFil: Bevacqua, Romina Jimena. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernandez Martin, R.. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal; ArgentinaFil: Pereyra Bonnet, Federico Alberto. Hospital Italiano; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cueto, M.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche; ArgentinaFil: Bruno Galarraga, María Macarena. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Patagonia Norte. Estación Experimental Agropecuaria San Carlos de Bariloche; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte; ArgentinaFil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Simple gene transfer technique based on I-SceI meganuclease and cytoplasmic injection in IVF bovine embryos

Although transgenic methods in mammals are inefficient, an easy and highly efficient transgenesis systemusing I-SceI meganuclease (intron-encoded endonuclease fromS. cerevisiae)was recently described in Xenopus. The method consisted of injection into fertilized eggs of an I-SceI reaction mixture with a plasmid DNA carrying the transgene, flanked by the meganuclease recognition sites (pIS). In the present study, the effects of I-SceI on gene transfer were tested apparently for the first time in mammals, in particular, in cattle. Various conditions were evaluated, including three concentrations of the plasmid pIS Pax6egfp, carrying I-SceI recognition sites flanking egfp under Pax6 promoter and two injection times (before IVM and after IVF) of pIS CAGegfp, carrying I-SceI sites fanking egfp under CAG promoter. In addition, the quantity of transgenewasmeasured using quantitative polymerase chain reaction, and presence of transgene signals was evaluated using fluorescence in situ hybridization analysis. Transgene expression rateswere higher (P< 0.05) for groups treated after IVF (79.1%, 91/115 and 63.0%, 75/ 119) than before IVM (32.6%, 31/95 and 34.7%, 33/95), with and without I-SceI, respectively. Interestingly, injectionwith pIS plus I-SceI after IVF increased frequency (P<0.05) of nonmosaic transgene-expressing embryos (58.3%, 42/72 vs. 29.7%, 25/84) for pIS plus I-SceI and pIS alone. Based on fluorescence in situ hybridization analysis, injectionwith I-SceI increased (P<0.05) the proportion of embryos with transgene signals in all blastomeres compared with pIS alone (44.0%,11/25 vs. 6.9%, 2/29) for pIS plus I-SceI and pIS alone. In addition, transgene copy number was numerically higher for the group treated with pIS plus I-SceI compared with pIS alone. In conclusion, I-SceI gene transfer increased transgene signals in bovine embryos.Fil: Bevacqua, Romina Jimena. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; ArgentinaFil: Canel, Natalia Gabriela. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; ArgentinaFil: Hiriart, María Inés. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Sipowicz, P.. Universidad Nacional de San Martín. Laboratorio de Neuro y Citogenética Molecular; ArgentinaFil: Rozenblum, G. T.. Universidad Maimónides. Area de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; ArgentinaFil: Vitullo, Alfredo Daniel. Universidad Maimónides. Area de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; ArgentinaFil: Radrizzani Helguera, Martin. Universidad Nacional de San Martín. Laboratorio de Neuro y Citogenética Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; ArgentinaFil: Fernández y Martín, Rafael. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; ArgentinaFil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentin

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 egfp–liposome 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.Fil: Salamone, Daniel Felipe. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Unidad Ejecutora de Investigaciones En Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones En Producción Animal; ArgentinaFil: Bevacqua, Romina Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Pque. Centenario. Unidad Ejecutora de Investigaciones En Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones En Producción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; ArgentinaFil: Pereyra Bonnet, Federico Alberto. Hospital Italiano; ArgentinaFil: Gambini, Andres. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal. Cátedra de Fisiología Animal; ArgentinaFil: Canel, Natalia Gabriela. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal. Cátedra de Fisiología Animal; ArgentinaFil: Hiriart, María Inés. Universidad de Buenos Aires. Facultad de Agronomia. Departamento de Producción Animal. Cátedra de Fisiología Animal; ArgentinaFil: Vichera, Gabriel Damian. Universidad Nacional de San Martín; ArgentinaFil: Moro, Lucía Natalia. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Jarazo, Javier

DNA fragmentation, transgene expression and embryo development after intracytoplasmic injection of DNA-liposome complexes in IVF bovine zygotes

Summary This study was designed to evaluate the quality and viability of bovine embryos produced by in vitro fertilization (IVF), after intracytoplasmic injection of pCX-EGFP-liposome complexes or pBCKIP2.8-liposome complexes (plasmids that codify the human insulin gene). Cleavage, blastocysts and expanded blastocysts rates of these both groups were not different from that of controls (IVF or IVF embryos injected with liposomes alone; IVF-L). The percentage of EGFP-positive (EGFP+) blastocysts was 41.8%. In Experiment 2, the blastocysts obtained after injection of pCX-EGFP-liposome complexes that did or did not express the transgene, were analyzed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labelling) assay at days 6, 7 and 8 of culture in vitro(Bd6, Bd7 and Bd8), in order to evaluate DNA fragmentation. The EGFP+ blastocysts showed different proportions of TUNEL-positive cells (T+) at Bd6, Bd7 and Bd8 (91, 73.7 and 99.5%, respectively) while blastocysts without EGFP expression (EGFP-) showed statistically lower numbers of fragmented nuclei (0, 44.6 and 85%, respectively; P < 0.05). There was no evidence of DNA fragmentation in either Bd6 or Bd7 IVF and IVF-L control blastocysts, but T+ nuclei were detected at Bd8 in both groups (66.4 and 85.8% respectively). Finally, IVF blastocysts (n = 21) injected with insulin-liposome complexes, cultured for 6, 7 and 8 days, were transferred to recipient cows. Pregnancy rates of 18.2% (2/11) and 40% (2/5) resulted from the transfer of Bd6 and Bd7 cells, respectively. Two pregnancies developed to term but they were not transgenic for the insulin gene. In conclusion, EGFP expression affects DNA integrity but not embryo development. Moreover, additional transfers are required in order to overcome the drawbacks generated by in vitro culture length and transgene expression.Fil: Vichera, G.. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Moro, Lucía Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentina. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Buemo, C.. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; ArgentinaFil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentin

Improved embryo development using high cysteamine concentration during IVM and sperm co-culture with COCs previous to ICSI in bovine

In contrast to other species, intracytoplasmic sperm injection (ICSI) in bovine remains inefficient, resulting in low embryo developmental rates. It is unclear whether such inefficiency is due to the poor response of bovine ooplasms to the injection stimulus, or to the inability of bull sperm to induce oocyte activation. In order to facilitate these events, two strategies were assessed: the use of high concentration of cysteamine [Cys] during IVM; and the selection of sperm attached to cumulus cells after incubation with COCs for ICSI. First, COCs were IVM with increasing [Cys] and subjected to IVF. Zygotes from all groups were cultured under different O2 tensions and development to blastocyst was evaluated. In a second experiment, sperm were co-cultured for 3 h with COCs and acrosome reaction was studied. Afterwards, the best IVM and IVC conditions determined on Experiment 1 were used for ICSI assay. COCs were matured for 21 h with 1 (Cys 1) or 0.1 mM Cys (Cys 0.1 groups, standard condition). In addition, COCs were incubated for ≥3 h with 16 × 106 sperm/ml and only sperm attached to cumulus cells were selected for ICSI (ICSI + Co-cult groups). After chemical activation, embryos were cultured in SOF medium under low O2 tension. Cleavage and blastocyst rates were evaluated at days 2 and 7 of IVC, respectively. Finally, the relative expression of eight genes indicators of embryo quality was compared between ICSI and IVF control blastocysts by qPCR. Cleavage rates were higher for Cys 0.1 ICSI + Co-cult and Cys 1 ICSI + Co-cult groups (n = 117, 92% and n = 116, 79%, respectively) compared to their controls (n = 132, 60% for Cys 0.1 ICSI and n = 108, 52% for Cys 1 ICSI) (p ≤ 0.05). Interestingly, the combined treatment (Cys 1 ICSI + Co-cult) showed higher blastocyst rates than all other ICSI groups (23 vs. 11, 18 and 14% for Cys 0.1 ICSI + Co-cult, Cys 1 ICSI, and Cys 0.1 ICSI, respectively) (p ≤ 0.05). Moreover, incubation with COCs increased the rates of live acrosome reacted sperm (p ≤ 0.05). The relative abundance of mRNAs coding for INFτ CAT, DNMT1, OCT4, and HDAC3 did not differ between treatments (p ≤ 0.05). SOD2, HADC1 and HADC2 expression was higher for Cys 0.1 ICSI than for IVF embryos (p ≤ 0.05). Group Cys 1 ICSI did not differ from IVF for those three genes, neither did Cys 1 ICSI + Co-cult, except for HDAC1 (p ≤ 0.05). In conclusion, the use of 1 mM Cys during IVM and of sperm incubated with mature COCs might be a good strategy to improve ICSI outcomes in cattle.Fil: Canel, Natalia Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones en Producción Animal; ArgentinaFil: Suvá, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones en Producción Animal; ArgentinaFil: Bevacqua, Romina Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones en Producción Animal; ArgentinaFil: Arias, María Elena. Universidad de La Frontera; ChileFil: Felmer Dörner, Ricardo Nicolás. Universidad de La Frontera; ChileFil: Salamone, Daniel Felipe. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones en Producción Animal; Argentin