Joaquim Mansano Garcia - Academia.edu (original) (raw)

Papers by Joaquim Mansano Garcia

Animal reproduction, 2015

Cryopreservation of oocytes is a strategic tool in embryo IVP but with limited use due to the com... more Cryopreservation of oocytes is a strategic tool in embryo IVP but with limited use due to the complex cellular structure of oocytes, being oocyte quality a factor that influences the success of the technique. In view of the role of IVM on oocyte quality, Simulated Physiological System Oocyte Maturation (SPOM; Albuz, Hum Reprod, vol 25, p 12; 2010), which utilizes cAMP modulators to achieve greater oocyte competence by the extension of meiosis block, was developed. The aim of this study was to investigate the effect of SPOM system on nuclear maturation and cytoskeletal integrity of vitrified bovine oocytes. Oocytes from slaughterhouse ovaries were divided into 8 groups: G1 (immature oocytes); G2 (matured in standard medium without FCS / 24 h); (G3 subjected to pre-IVM / 2 h in the presence of modulators of cAMP, Forskolin (100μM) and IBMX (500μM), and then the extended IVM / 28 h with Cilostamide (20μM) and FSH); G4 (immature oocytes vitrified and subjected to conventional IVM); G5 (...

Biology of Reproduction, 2007

ABSTRACT The production of suitable cytoplasts for cloning trough nucleartransfer, demand the rem... more ABSTRACT The production of suitable cytoplasts for cloning trough nucleartransfer, demand the remove of the chromosomes in the MetaphaseII plate (enucleation) without damage the oocyte. Enucleationof the oocyte can be performed mechanically or chemically. Mechanicalenucleation can damage the oocyte due to the removal of a largeamount of cytoplasm together with the chromosomes. Moreoverfor mechanical enucleation is necessary to stain the chromatinwith a fluorescent dye and use an inverted fluorescent microscope,with made the technique more expensive. Chemical enucleationis an alternative, since it minimize the amount of cytoplasmremoved and eliminate the use of fluorescent staining. For chemicalenucleation several drugs can be used as colchicine and demecolcine.The demecolcine impair the organization of the microtubulesleading to disruption of the metaphase plate. Metaphase II oocytestreated with demecolcine presents a protrusion on the corticalregion of the oocytes containing the chromosomes. Although theuse of demecolcine has been reported in domestic animals suchas cows and pigs, in horses there is no report concerning chemicalenucleation methods. The aim of this experiment was to studythe use of demecolcine (Sigma, D1925) for chemical enucleationof equine oocytes destined to nuclear transfer. Equine and bovineoocytes were obtained from slaughterhouse ovaries and maturedin 5% CO2 in air at 38. 5° C. For in vitro maturation (IVM)equine oocytes were cultured in 4 well dishes with 400µlHTF:BME (1:1) media with 0.3% BSA, 0.32mM pyruvate, 1mM L-glutamine,0.4mM cisteine, 0.1mM taurine, 0.4mM glicine, 3.3mM sodium lactate,100ng/ml IGF-1, 50ng/ml EGF, 100ng/ml eGH, 5µg/ml eFSH(Bioniche), 500ng/ml estradiol and 75µg/ml gentamicin.Bovine oocytes were matured in TCM 199 + 10% FCS, 2.2 mg/mlpyruvate, 1mg/ml estradiol, 50µg/ml hCG, 5µg/mlFSH and gentamicin. Nuclear maturation was estimated by stainingthe oocytes with Hoescht 33342 at the end of maturation period.For chemical enucleation demecolcine was used to induce theprotrusion of the metaphase plate allowing the remove of thechromosomes without staining the DNA. After 30 and 20 hoursof IVM equine and bovine oocytes, respectively, were stripedand treated with 0.2 µg/mL demecolcine for equine and0.05µg/mL for bovine for 2 hours. The oocytes were thenwashed 3 times in MEM + 10% FCS and selected for the presenceof the 1° polar body (PB) and the protrusion in an invertedmicroscope (Leica- DMIRB). Three replicates were made with 45oocytes/group/replicate. The confirmation of nuclear materialinside the protrusion was confirmed by staining the oocyteswith Hoescht 33342. The maturation rates (MII) were 65% forbovine oocytes, 36% for expanded equine oocytes and 25% forcompact equine oocytes. After demecolcine treatment 38.7% ofthe bovine oocytes presented a visible PB associated with aprotrusion. The detection of the protrusion was possible in23.6% and 28.3% for expanded and compact equine oocytes respectively.The results showed that, although enucleation rates of equineoocytes using demecolcine were lower than the bovine, the techniqueis a viable method for preparing oocytes for nuclear transfer.However, a dose response study needs to be done aiming the improvementof enucleation rates. Acknowledgements: FAPESP (Grant 04/00822–1),Santa Fé Slaughterhouse and Dr. Gercio Bonesi (poster)

Ciência Animal Brasileira, Jul 3, 2009

Reproduction, Fertility and Development, 2008

The developmental competence of enucleated oocytes is a key factor that determines the overall su... more The developmental competence of enucleated oocytes is a key factor that determines the overall success of animal cloning. Enucleation is an invasive procedure in traditional nuclear transfer (NT). The objective of this work was to evaluate the effects of demecolcine, a microtubule-depolymerizing agent, on metaphase II (MII) bovine oocytes and to verify the capacity of embryonic development after NT using chemically assisted enucleation. In the first experiment, oocytes after 21 h of IVM were exposed for 2 h to several concentrations of demecolcine: 0 (control), 0.025, 0.05, 0.2, and 0.4 µg mL–1, and evaluated in relation to membrane protrusion formation. After the best concentration of demecolcine was determined, the nuclear and microtubular dynamics of the treated oocytes were evaluated by immunofluorescence microscopy of tubulin and chromatin (Liu et al. 1998 Biol. Reprod. 5, 537–545) in a second experiment. The results were analyzed by Duncan and Tukey tests in Experiments I and ...

Arquivo Brasileiro de Medicina Veterinária e Zootecnia, 2003

ABSTRACT O objetivo deste trabalho foi avaliar as taxas de ativação e de clivagem de oócitos bovi... more ABSTRACT O objetivo deste trabalho foi avaliar as taxas de ativação e de clivagem de oócitos bovinos tratados com estrôncio (10mm de SrCl2), após maturação in vitro por 27-28 horas. No experimento 1, os tratamentos foram: S4 - ativação pelo estrôncio por 4 horas; S12 - ativação pelo estrôncio por 12 horas; S30 - ativação pelo estrôncio por 30 horas; e P - ativação por pulso elétrico (3 pulsos de 1,0kv/cm). No experimento 2 os tratamentos foram: PS4 - ativação combinada pelo pulso elétrico e pelo estrôncio por 4 horas; S4P - ativação pelo estrôncio por 4 horas e pelo pulso elétrico; e PS30 - ativação pelo pulso elétrico e pelo estrôncio por 30 horas. No experimento 1, todos os tratamentos apresentaram taxas similares de ativação (83-90%; P>0,05). Para clivagem, P foi melhor (53%; P<0,05) do que todos os tratamentos com estrôncio (6 a 28%). No experimento 2, o tratamento S4P apresentou melhor taxa de ativação (88%; P<0,05) do que PS4 e PS30 (60 e 68%, respectivamente). Para clivagem, observou-se o mesmo padrão, S4P (65%; P<0,05) e PS4 e PS30 (37% e 44%, respectivamente). Conclui-se que o estrôncio é capaz de ativar oócitos bovinos e sua combinação com pulso elétrico não melhora a ativação. Este é o primeiro relato demonstrando que o estrôncio ativa oócitos bovinos.

Arquivo Brasileiro de Medicina Veterinária e Zootecnia, 2001

The effects of insulin-like growth factor-I (IGF-I) on in vitro maturation (IVM) (experiment I) a... more The effects of insulin-like growth factor-I (IGF-I) on in vitro maturation (IVM) (experiment I) and on in vitro embryo development (experiment II) of bovine oocytes fertilized in vitro, were evaluated in terms of cleavage (CR), blastocyst (BR) and hatching (HR) rates. For IVM, immature cumulus-oocyte complexes were cultured in TCM-199 medium supplemented with Hepes, sodium bicarbonate, sodium pyruvate, additives, fetal calf serum (B-199 medium) and gonadotropins (14 U/ml PMSG and 7 U/ml hCG). For embryo development, the oocytes/zygotes were cultured in B-199 medium with bovine oviduct epithelial cells in suspension under silicon oil. Treatments for in vitro culture conditions for both experiments were: 1- B-199 + 200 ng/ml IGF-I; 2- B-199 + 100 ng/ml IGF-I; 3- B-199 + 50 ng/ml IGF-I; 4- B-199 + 10 ng/ml IGF-I; 5- B-199 + 0 ng/ml IGF-I. All cultures were performed at 38.5ºC in 5% CO2 in air and the data were analyzed by chi-square test. In experiment I, there were no differences (P&g...

Animal Reproduction Science, 2004

Current trends in biomedical engineering & biosciences, Apr 29, 2021

Revista Brasileira De Zootecnia-brazilian Journal of Animal Science, Apr 1, 2000

Journal of Visualized Experiments, Dec 16, 2018

Reproduction, Fertility and Development, 2014

Agrarian, 2013

Para se alcançar maiores índices reprodutivos em cabras, o fotoperíodo artificial tem sido usado ... more Para se alcançar maiores índices reprodutivos em cabras, o fotoperíodo artificial tem sido usado apenas na sincronização de estro. Além disso, esta espécie animal tem importância na produção de animais transgênicos produzidos por FIV. O objetivo deste estudo foi produzir e comparar embriões caprinos (Capra hircus-Linnaeus-1758) provenientes de cabras Alpinas submetidas ao fotoperíodo natural (estação e anestro) e artificial (120 luxes/45 dias/20 h). Foram utilizadas 31 cabras Alpinas, distribuídas em cinco grupos, avaliando o fotoperíodo natural e artificial, utilizando sêmen fresco e congelado e heparina (200μg mL-1) na capacitação espermática. Foram obtidos 720 oócitos e sete blastocistos (cinco do G3- fotoperíodo natural e dois no G2 –fotoperíodo artificial, ambos durante o anestro estacional). Estes foram congelados por um ano. Ao término do estudo concluiu-se que não houve diferença estatística entre os grupos. Após a descongelação e transferência de sete embriões, obteve-se o...

Ciência Animal Brasileira, 2009

It has been the aim to value, in Nelore cows, the association of the assisted reproduction MOET e... more It has been the aim to value, in Nelore cows, the association of the assisted reproduction MOET e OPU-PIV. For this, it has tested the ovarian superovulation (SOV) 48 to 60 h after the OPU (ovum pick-up) at a random phase of the estral cycle (OPU1) and the in vitro production (PIV) of recovered oocytes embryos (OPU2) in different moments after the prostaglandin F2? (PGF2?) application. The females (n=23) after the OPU1 received progestogen implant and 48 to 60 h after the OPU, then it has began the SOV, with FSH 180 mg / cows in 8 doses, for 4 days. In 6th and 7th applications of FSH, there were administered 500 µg of PGF2? being the implants withdrawn in the 7th dose. After 12 h of the SOV, it was applied 25mg of LH to realize AI (artificial insemination) with fixed time. In the day of the embryos recovery 6 treatments were tested: T (0-144) (n=4), T (48-120) (n=5), T (48-96) (n=4), T (72-96) (n=3), T (96-72) (n=4), T (96-48) (n=3) which varied in accordance with moments of the PG...

Genetics, 2001

Due to the exclusively maternal inheritance of mitochondria, mitochondrial genotypes can be coupl... more Due to the exclusively maternal inheritance of mitochondria, mitochondrial genotypes can be coupled to a particular nuclear genotype by continuous mating of founder females and their female offspring to males of the desired nuclear genotype. However, backcrossing is a gradual procedure that, apart from being lengthy, cannot ascertain that genetic and epigenetic changes will modify the original nuclear genotype. Animal cloning by nuclear transfer using host ooplasm carrying polymorphic mitochondrial genomes allows, among other biotechnology applications, the coupling of nuclear and mitochondrial genotypes of diverse origin within a single generation. Previous attempts to use Bos taurus oocytes as hosts to transfer nuclei from unrelated species led to the development to the blastocyst stage but none supported gestation to term. Our aim in this study was to determine whether B. taurus oocytes support development of nuclei from the closely related B. indicus cattle and to examine the fa...