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Ureaplasma diversum pode causar infecções e ativar a resposta imune, estando relacionado com infertilidade em bovinos. Devido à importância dos macrófagos nessas infecções e a relação direta com blastocistos bovinos, o estudo teve o objetivo avaliar o perfil imune in vitro destas células após a infecção experimental por U. diversum. Demonstrou-se capaz de promover resposta inflamatória em macrófagos murinos e bovinos e blastocistos bovinos, com ativação do TLR2 e secreção de IL-1β e TNF-α. A ativação inflamatória pode estar relacionada com a presença de um componente de superfície capaz de induzir uma resposta desse tipo, como lipoproteínas associadas aos lipídeos de membrana (LAMPs), mas as de U. diversum ainda não foram totalmente caracterizadas. Estes resultados associados ao sequenciamento do genoma de U. diversum, permite avanço na compreensão da biologia molecular de infecções por micoplasma. Assim, fomenta-se a discussão da importância e relevância das micoplasmoses, especialmente por U. diversum, na reprodução e bovinocultura no cenário brasileiro. / Ureaplasma diversum can cause infection and activate the immune response, and is associated with infertility in cattle. Due to the importance of macrophages in these infections and the direct relationship with bovine blastocysts, the study aimed to evaluate the immune profile in vitro of these cells after experimental infection U. diversum. It has been shown capable of promoting inflammatory response in murine and bovine macrophages and bovine blastocysts, with activation of TLR2 and IL-1β secretion and TNF-α. The inflammatory activation may be related to the presence of a surface component capable of inducing a response of that type, such as lipoproteins associated with lipid membrane (LAMP\'s), but U. diversum lipoproteins have not been fully characterized. These results associated with the sequencing of the U. diversum genome allows improvement in understanding the molecular biology of mycoplasma infections. Thus, encourages the discussion of the importance and relevance of mycoplasmosis, especially U. diversum, reproduction and cattle in the Brazilian scene.

Resumo: Efeitos deletérios dos radicais livres causam a lipoperoxidação da membrana plasmática dos gametas feminino e masculino e dos embriões cultivados in vitro, sendo responsável por perda da produção in vitro de embriões e redução da fertilidade in vivo. O estudo foi realizado com objetivo de investigar o efeito protetor do diluidor de sêmen contendo antioxidante submetido à criopreservação e avaliado por testes complementares (teste de termo resistência, hiposmótico e fluorescência), pela inseminação artificial e fertilização in vitro. Ejaculados de cinco touros foram agrupados nos seguintes tratamentos: 1 - Diluidor Tris - gema (controle - Dil C); 2 - Diluidor Tris -gema + Trolox 200mM (antioxidante-Dil T). Amostras com os diluidores com concentração final de 25 x 106 espermatozóides/ palheta foram envasadas em palhetas de 0,25 mL. Em uma fazenda localizada no Mato Grosso do Sul - MS, foram inseminadas 300 novilhas Nelore, e para cada touro realizou-se 60 inseminações artificiais (palhetas descongeladas em 35°C por 20 seg). Oócitos com citoplasma homogêneo e cumulus compacto de ovário de vacas de abatedouro foram selecionados e maturados em grupos de 25 por gota em 100μl de meio TCM 199 com SFB, FSH, hCG e estradiol, piruvato de sódio e amicacina, por 24 horas, sob óleo mineral, e foram cultivados com 5% CO2 e 95% umidade em ar, a 38,5ºC. Depois da maturação, os oócitos foram colocados em gotas com TALP contendo BSA, PHE e 10μg/ml de heparina com 1x 106 espermatozóides móveis por ml. Quatro palhetas (duas - DC e duas - DT) do mesmo touro e ejaculado foram descongeladas e cada palheta foi processada por um método de separação espermática para recuperação do sobrenadante: método de lavagem (duas centrifugações de 36xg por 5 min em 2ml de TL-sêmen) e gradiente de Percoll (45 e 90%, submetido a centrifugação a 500xg por 30 min)...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Deleterious effects of free radicals cause lipoperoxidation of the plasmatic membrane of female and male gametes and in vitro cultured embryos, being responsible for losses in in vitro production of embryos and reduction in in vivo fertility. The objective of this study was to investigate the protective effects of semen extender containing antioxidant submitted to cryopreservation and evaluated by artificial insemination, in vitro fertilization and complementary tests (hyposmotic, fluorescence, and thermal resistance tests). Ejaculates from 5 bulls were treated with Tris egg yolk extender (control-CE) alone or supplemented with 200ml Trolox in the extender (antioxidant-AE). The samples with the extenders with a final concentration of 25 x 106 spermatozoa/ml were placed into 0.25 ml straws. Three hundred Nelore heifers from a cattle farm in Mato Grosso do Sul - MS, were separated into 5 groups of 60 animals. For artificial insemination, the straws were thawed at 35°C for 20 sec and the females in each group were inseminated with semen of the same bull, using 30 female for each semen extender treatment. For in vitro fertilization, oocytes with homogeneous cytoplasm and compact cumulus, collected from ovaries of slaughtered cows were selected and maturated in groups of 25 in droplets of 100μl TCM 199 medium with FCS, FSH, hCG and estradiol, sodium pyruvate and amicacin, for 24 hours, under mineral oil, in atmosphere of 5% CO2 and 95% humidity in air, at 38.5ºC. After maturation, the oocytes were placed in droplets with TALP containing BSA, PHE and 10μg/ml of heparin with 1x 106 motile spermatozoa/ml...(Complete abstract, click electronic access below) / Orientador: Paulo Henrique Franceschini / Coorientador: Antônio Cláudio Tedesco / Banca: Joaquim Mansano Garcia / Banca: César Roberto Esper / Banca: Sony Dimas Bicudo / Banca: José Domingos Guimarães / Doutor

The extraembryonic endoderm (XEN) defines the yolk sac, a set of membranes that provide essential support for mammalian embryos. Recently, the committed XENprecursor was identified in the embryonic Inner Cell Mass (ICM) as a group of cells that intermingles with the closely related, anatomically indistinguishable epiblast (EPI)- precursor that gives rise to the fetus. In vitro, the EPI-precursor is represented by the well-known embryonic stem (ES) cell lines, but cell lines representing the XENprecursor are not known. Furthermore, since the XEN-precursor cells were discovered only very recently, the unexpected fact that they express the key pluripotency marker Oct4 has not been explored. Recently, however, our laboratory has isolated rat XEN cell lines that express Oct4, leading to the following two questions: (i) Do these new XEN cell lines represent XEN-precursor cells? (ii) Is their Oct4 expression regulated similarly as previously known from ES cells? These two questions are addressed here by lineage marker and reporter gene analyses. Whole culture analyses showed that rat XEN cell lines expressed markers of all XEN stages including XEN-precursor, primitive endoderm (PrE) and/or visceral endoderm (VE), and parietal endoderm (PE) but trophoectoderm and EPI-precursor markers were missing. In line with this, immunocytochemistry demonstrated heterogeneity and directly visualized the XEN-precursor, PrE/VE, and PE subpopulations. Low-density plating and time-dependent immunocytochemistry on resulting colonies strongly suggested that XEN-precursor cells generate the other XEN stages. Moreover, by analyzing single-cell derived clones, it was shown that culture heterogeneity results from the self-renewal and differentiation of a single cell. Reporter gene analyses using the 5’ regulatory region of the mouse Oct4 gene revealed that a DNA fragment containing the previously described distal enhancer drove reporter gene expression only in ES cells whereas inclusion of an upstream fragment led to high expression in both mouse ES and rat XEN cells. In conclusion, our rat XEN cell lines contain XEN-precursor cells that differentiate extensively, providing for the first time an in vitro model that mimics the natural process of early XEN differentiation. In addition, they regulate Oct4 gene transcription differently than ES cells suggesting heterogeneous Oct4 regulation within the mammalian ICM.

Made available in DSpace on 2014-06-11T19:35:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-02-27Bitstream added on 2014-06-13T20:06:42Z : No. of bitstreams: 1 borges_jc_dr_jabo.pdf: 527084 bytes, checksum: 74a9732f86335b470c5edaba6623feba (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Efeitos deletérios dos radicais livres causam a lipoperoxidação da membrana plasmática dos gametas feminino e masculino e dos embriões cultivados in vitro, sendo responsável por perda da produção in vitro de embriões e redução da fertilidade in vivo. O estudo foi realizado com objetivo de investigar o efeito protetor do diluidor de sêmen contendo antioxidante submetido à criopreservação e avaliado por testes complementares (teste de termo resistência, hiposmótico e fluorescência), pela inseminação artificial e fertilização in vitro. Ejaculados de cinco touros foram agrupados nos seguintes tratamentos: 1 – Diluidor Tris – gema (controle – Dil C); 2 – Diluidor Tris –gema + Trolox 200mM (antioxidante-Dil T). Amostras com os diluidores com concentração final de 25 x 106 espermatozóides/ palheta foram envasadas em palhetas de 0,25 mL. Em uma fazenda localizada no Mato Grosso do Sul – MS, foram inseminadas 300 novilhas Nelore, e para cada touro realizou-se 60 inseminações artificiais (palhetas descongeladas em 35°C por 20 seg). Oócitos com citoplasma homogêneo e cumulus compacto de ovário de vacas de abatedouro foram selecionados e maturados em grupos de 25 por gota em 100μl de meio TCM 199 com SFB, FSH, hCG e estradiol, piruvato de sódio e amicacina, por 24 horas, sob óleo mineral, e foram cultivados com 5% CO2 e 95% umidade em ar, a 38,5ºC. Depois da maturação, os oócitos foram colocados em gotas com TALP contendo BSA, PHE e 10μg/ml de heparina com 1x 106 espermatozóides móveis por ml. Quatro palhetas (duas – DC e duas – DT) do mesmo touro e ejaculado foram descongeladas e cada palheta foi processada por um método de separação espermática para recuperação do sobrenadante: método de lavagem (duas centrifugações de 36xg por 5 min em 2ml de TL-sêmen) e gradiente de Percoll (45 e 90%, submetido a centrifugação a 500xg por 30 min)... / Deleterious effects of free radicals cause lipoperoxidation of the plasmatic membrane of female and male gametes and in vitro cultured embryos, being responsible for losses in in vitro production of embryos and reduction in in vivo fertility. The objective of this study was to investigate the protective effects of semen extender containing antioxidant submitted to cryopreservation and evaluated by artificial insemination, in vitro fertilization and complementary tests (hyposmotic, fluorescence, and thermal resistance tests). Ejaculates from 5 bulls were treated with Tris egg yolk extender (control-CE) alone or supplemented with 200ml Trolox in the extender (antioxidant-AE). The samples with the extenders with a final concentration of 25 x 106 spermatozoa/ml were placed into 0.25 ml straws. Three hundred Nelore heifers from a cattle farm in Mato Grosso do Sul - MS, were separated into 5 groups of 60 animals. For artificial insemination, the straws were thawed at 35°C for 20 sec and the females in each group were inseminated with semen of the same bull, using 30 female for each semen extender treatment. For in vitro fertilization, oocytes with homogeneous cytoplasm and compact cumulus, collected from ovaries of slaughtered cows were selected and maturated in groups of 25 in droplets of 100μl TCM 199 medium with FCS, FSH, hCG and estradiol, sodium pyruvate and amicacin, for 24 hours, under mineral oil, in atmosphere of 5% CO2 and 95% humidity in air, at 38.5ºC. After maturation, the oocytes were placed in droplets with TALP containing BSA, PHE and 10μg/ml of heparin with 1x 106 motile spermatozoa/ml...(Complete abstract, click electronic access below)

The mammalian embryo is encased in a glycoproteinaceous covering, the zona pellucida (ZP/zona) during preimplantation development. Prior to implantation into the recipient maternal endometrium, the blastocyst has to hatch out of this zona. This is a critical and an important event for the successful establishment of pregnancy. Hatching in mammals is characterised by the expansion of the blastocyst, followed by the nicking of the zona and extrusion of the blastocyst by repeated contraction-expansion cycles, thereby leaving the empty zona behind. In species such as the mouse, cow and primates, the empty zona is left behind in the uterine lumen. However, in the hamsters, the features associated with hatching are characteristic for this species. Firstly, the blastocyst remains predominantly in a deflated state. Secondly, the zona undergoes focal rupture which is followed by the complete dissolution of the zona. Third, trophectodermal projections (TEPs) present in the blastocysts, aid the hatching of the blastocyst. Hence, this study was aimed to identify the molecular players involved in hamster blastocyst hatching and to study their embryo-endometrial expression. Earlier work in the laboratory has demonstrated the involvement of cysteine protease-like factors in hamster blastocyst hatching (Mishra and Seshagiri, 2000a). Broad spectrum cysteine protease inhibitors, E-64 and PHMB, completely blocked the hatching of blastocysts. To identify the class of cysteine proteases involved in this phenomenon, class-specific inhibitors were used in this study. Calpain and caspase inhibitors, calpastatin and Z-VAD- FMK, respectively, did not block hamster blastocyst hatching (Fig 2.2). Cathepsin (cts)-specific inhibitors, cystatin-C and peptidyl diazomethane (PPDM) blocked hatching of embryos in a dose-, time- and embryo-stage dependant manner (Figs 2.5, 2.6, 2.10 and 2.11). Continuous exposure of 1.0 µM cystatin to expanded or deflated blastocysts completely blocked hatching (Fig 2.3Aii, iii), without effecting their viability (Fig 2.3Bii and iii). Deflated blastocysts exposed transiently to 1.0 µM cystatin, for 12 or 6 h failed to hatch, but could overcome the inhibition and exhibited hatching, when transferred to fresh, inhibitor-free medium (Fig 2.6). Effect of the inhibitor was less pronounced in the deflated blastocysts when compared to expanded blastocysts (Figs 2.5 and 2.6). The viability of the cystatin-treated embryos was not affected as assessed by vital-dye staining and also their ability to attach and exhibit trophoblast (TB) proliferation on serum-coated dishes was not compromised. The area of the TB outgrowth of cystatin-treated embryos was similar to that of the untreated embryos (Fig 2.9). The inhibitory effect of PPDM, an irreversible inhibitor of cts, on blastocyst hatching was demonstrated. Expanded and deflated blastocysts exhibited a dose-dependent inhibition in hatching, following the exposure to 0.5 and 1.0 µM of PPDM (Figs 2.10A and 2.11A). When treated with the inhibitor for 6 or 12 h, there was a transient inhibition in hatching, as blastocysts could overcome the inhibition and exhibit hatching following transfer to inhibitor-free, fresh medium. Inhibitor-treated hatched blastocysts, when transferred to serum-coated dishes, attached and exhibited TB outgrowth, similar to untreated embryos (Figs 2.13 and 2.14). A PPDM-interacting protease was localised to the cytoplasm of the embryonic cells in the hamster blastocyst, suggesting that the embryo is the source of the zona lysin. Two forms of the enzyme, a probable variant zymogen of molecular mass 65 k and an active form of molecular mass 32 k were detected in the blastocysts (Fig 2.15). In vitro susceptibility of hamster zona to cathepsins is significantly different from that of other species zonae such as the mouse, rat, monkey and human zonae (Table 2.2). All these lines of evidence unequivocally demonstrate the involvement of cathepsins in hamster blastocyst hatching, which is in sharp contrast to what is observed in the mouse, where serine proteases such as strypsin/hepsin, ISP-1 and -2 are reported to play an important role in blastocyst hatching. However, since extensive inhibitor studies were not performed using embryos from other species, it is possible that cysteine proteases maybe involved in the hatching of blastocysts from other species. Having shown the role of cathepsins in hamster zona dissolution, expression of the cathepsins in preimplantation embryos was investigated. Hamster specific cts–L, -B and –P were amplified from day 14 placenta using mouse primers and the amplicons were found to be highly homologous to the cts of other species (Fig 3.2). Hamster and mouse preimplantation embryos i.e., 8-cell, morula and blastocyst were found to express cts–L, -B and –P transcripts (Figs 3.6 and 3.10). Cts-P, present only in the TBs of the placenta (Fig 3.4), for the first time, was also shown to be present in the preimplantation embryos. The immunoreactive cts-L and -P proteins were detected in blastomeres of 8-cell embryo, in the inner cell mass (ICM) and trophectoderm (TE) of the blastocyst (Figs 3.7 and 3.8). These cathepsins could probably correspond to the PPDM- interacting enzymes of molecular mass 32 and 65 kDa, described above. (fig) Fig 5.1. Overview of the expression and the role of embryo-endometrial cathepsins in blastocyst hatching in the golden hamster. Cathepsins ( ) produced by the inner cell mass ( ) or the trophectoderm ( ) of the blastocyst or the endometrial cells ( ) act on the zona matrix ( ), bringing about its lysis. The cathepsins are secreted into the peri-vitelline space or are carried by trophectodermal projections (TEPs, yellow projections) to the zona. Also shown are endogenous inhibitors and growth factors that can regulate these cathepsins. A striking observation made in this study was the detection of the immunoreactive signals for cathepsins in the zona matrix of blastocysts. Since hamster blastocysts possess extracellular projections (TEPs), it is possible for these projections to participate in the transport of cathepsins from TE cells to the zona; as the localisation of the proteases to these projections was demonstrated (Fig 3.9). Also, since the actin-based projections are highly undulating structures, they might potentiate the mechanical rupture of the zona during hatching, apart from acting as carriers for the proteases. Hence, during hatching of the hamster blastocyst, cathepsins, expressed in the ICM and the TE, might be secreted transiently into the peri-vitelline space, whereby they can act on the ZP. Alternatively, in the absence of any apoptotic cells in the embryo that can release the cell contents (Fig 3.13), the cathepsins may be deposited by TEPs in specific pockets of the zona matrix, thereby causing focal zona lysis. In vivo, the hatching of the blastocysts is brought about by both embryonic and maternal proteases. Cts–L and -B transcripts were detected in the maternal endometrium during different stages of the reproductive cycle and early pregnancy (Fig 4.1 and 4.3). Immunoreactive cts-L protein was detected in the uterine luminal epithelium and the stromal cells (Fig 4.5). In the uterus, the PPDM-interacting 32 kDa form was in abundance compared to the 65 kDa form (Fig 4.6). Hence, uterine cathepsins might play a major role in the remodelling of the extracellular matrix during estrous cycle and pregnancy. However, the role of these cathepsins in causing zona dissolution during blastocyst hatching, along with embryonic proteases cannot be ruled out. Reports of recurrent miscarriages in women with low serum cystatin levels imply a role for cysteine proteases in early pregnancy events like blastocyst development, hatching and implantation. Hence, these studies, described in the thesis, could form a basis to investigate the role of cathepsins in early human development. Taken together, the results demonstrate the involvement of embryo-derived cathepsins in hamster blastocyst hatching. These cathepsins may be secreted into the peri-vitelline space or transported to the zona matrix by TEPs (Fig 5.1). Additionally, in vivo, endometrial cathepsins might aid the embryonic zona lysins in the complete zona dissolution. The regulation of these proteases by growth factors, cytokines and their specific inhibitors needs to be explored. (For figure pl see the original document)

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-09-16T11:53:05Z No. of bitstreams: 1 ribrioivantavarespereirabatista.pdf: 1023205 bytes, checksum: 9149da7ed1e431e5565e4816dc74a814 (MD5) / Approved for entry into archive by Diamantino Mayra (mayra.diamantino@ufjf.edu.br) on 2016-09-26T20:21:30Z (GMT) No. of bitstreams: 1 ribrioivantavarespereirabatista.pdf: 1023205 bytes, checksum: 9149da7ed1e431e5565e4816dc74a814 (MD5) / Made available in DSpace on 2016-09-26T20:21:30Z (GMT). No. of bitstreams: 1 ribrioivantavarespereirabatista.pdf: 1023205 bytes, checksum: 9149da7ed1e431e5565e4816dc74a814 (MD5) Previous issue date: 2010-02-25 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A suplementação do ácido linoléico conjugado trans-10, cis-10 no meio de cultivo, representa uma importante alternativa para aumento da sobrevivência dos embriões após a criopreservação. No entanto este isômero de CLA no cultivo in vitro sem antioxidante pode aumentar a taxa apoptótica. O objetivo do presente estudo foi avaliar o efeito da adição CLA trans-10, cis-12 no cultivo in vitro de embriões sem antioxidante. Zigotos bovinos (n = 1.694) foram divididos em dois tratamentos: (T1) grupo controle, zigotos cultivados no meio CR2aa suplementado com soro fetal (n = 815); (T2) zigotos cultivados no meio CR2aa suplementado com soro fetal mais 100 µM CLA trans-10, cis-12. Os embriões foram avaliados quanto a desenvolvimento, quantidade de lípides e criotolerância. Transcritos dos RNA mensageiros (RNAm) dos genes selecionados foram mensurados pelo Real Time PCR. Suplementação de CLA trans-10, cis-12 não afeta significativamente a taxa de blastocisto (31,8% e 34,1% T1 e T2, respectivamente, p = 0,20) e nível dos RNAm dos genes relacionados com stress celular, apoptose e síntese de novo de ácido graxo. Quantidade de lípides e transcritos do RNAm do gene 1-acilglicerol-3-fosfato oaciltransferase 1 enzima relacionado a síntese de triglicérides foram significativamente reduzidos nos embriões cultivados na presença de CLA trans-10, cis-12 em comparação com o grupo controle. Teve aumento significativo na taxa de re-expansão dos blastocistos cultivados na presença de CLA trans-10, cis-12, após o descongelamento (34.4 e 56.3% para T1 e T2, respectivamente p = 0,002). Essa diferença não persistiu na taxa de eclosão (14,0% e 16,5% para T1 e T2, respectivamente, P = 0,62). Esses resultados mostram que o CLA trans-10, cis-12 reduz o acúmulo de lípides nos embriões pela redução nos níveis dos transcritos do gene 1-acilglicerol-3-fosfato o-aciltransferase 1 sem afetar a qualidade do embrião. Adicionalmente, este ácido graxo aumenta a taxa de re-expansão, no entanto, não melhora a taxa de eclosão. / Supplementation of conjugated linoleic acid trans-10, cis-10 in the culture medium, represents an important alternative to increasing the survival of embryos after cryopreservation. However the addition of culture media with CLA trans-10, cis-12 without antioxidant may increase the apoptotic rate. The aim of this study was to evaluate the effect of adding CLA trans-10, cis-12 in vitro culture of embryos without antioxidant. Bovine zygotes (n = 1,694) were divided into two treatments: (T1) control group, zygotes cultured in CR2aa medium supplemented with fetal calf serum (n = 815), (T2) zygotes cultured in CR2aa medium supplemented with fetal calf serum plus 100 µM CLA trans-10, cis-12. Embryos were evaluated for development, amount of lipids and cryotolerance. Transcripts of messenger RNA (mRNA) of selected genes were measured by real time PCR. Supplementation of CLA trans-10, cis-12 did not significantly affect the blastocyst rate (31.8% and 34.1% T1 and T2, respectively, p = 0,20) and the mRNA level of genes related to cell stress, apoptosis and de novo synthesis of fatty acid . Lipids and transcripts of the mRNA of the gene 1-acilglicerol-3-phosphate o-acyltransferase 1 enzyme related to the synthesis of triglycerides were significantly reduced in embryos cultured in the presence of CLA trans-10, cis-12 in comparison the control group. Had increased rate re-expansion of blastocysts cultured in the presence of CLA trans-10, cis-12, after thawing (34.4 and 56.3% for T1 and T2, respectively p = 0,002). This difference did not persist in the hatching rate (14.0 and 16.5% for T1 and T2, respectively, P = 0,62). These results show that the CLA trans-10, cis-12 reduces the accumulation of lipids in the embryos by reducing the levels of gene transcripts acilglicerol-1-3-phosphate oacyltransferase 1 without affecting the quality of the embryo. Additionally, this fatty acid increases the rate re-expansion, but does not improve the hatching rate.

En un intento de optimizar el sistema de fecundación in vitro en la especie porcina se estudió la influencia de distintas condiciones de maduración y de cocultivo de los gametos. Se evaluó la reducción del tiempo de coincubación de los gametos a 10 min observándose un claro efecto del ratio espermatozoides:ovocito. El estudio de las necesidades de los espermatozoides en términos de aditivos del medio de fecundación y tiempo de coincubación reveló variaciones entre verracos. La utilización de un tiempo de coincubación tan corto como 2 min fue suficiente para obtener unas tasas de penetración y monospermia similares a las alcanzadas por los sistemas de FIV tradicionales. El sistema de FIV en pajuela con 10 min de coincubación aumentó la penetración monoespérmica y mejoró la calidad de los blastocistos. La adición de 5 nM de 9- cis ácido retinoico al medio de maduración aumentó significativamente la formación de blastocistos. / The present study was conducted in an attempt to optimize porcine in vitro fertilization system. For this purpose, the influence of maturation and gamete coculture conditions were studied. The coincubation time may be reduced to 10 min to increase the efficiency of fertilization depending on the sperm:oocytes ratio. The needs of boar spermatozoa for IVF, in terms of additives to IVF medium and coincubation times vary among boars. The use of coincubation time as brief as 2 min is long enough to obtain good fertilization rates similar to those achieved from current long term exposure times in IVF. A straw IVF system in combination with a 10 min coincubation increased monospermic penetration and the quality of blastocysts compared with the microdrop-IVF system. The addition of 5 nM of 9- cis retinoic acid in the IVM medium increased blastocyst formation rate, suggesting that RA may play an important role during IVM.