Efeito das condições de cultivo no remodelamento das histonas de embriões bovinos produzidos in vitro

Pós-graduação em Medicina Veterinária - FCAV

Desenvolvimento de embriões in vitro na presença de IGF-1, GH e insulina nos meios de maturação de ovócitos e cultivo embrionário

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeito do soro fetal bovino e do etossulfato de fenazina sobre o acúmulo lipídico, apoptose e resposta à vitrificação em embriões bovinos produzidos in vitro

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Resultados da FIV/ICSI após o desenvolvimento do embrião humano em baixa concentração de oxigênio: uma meta-análise

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Avaliação dos efeitos da inibição da maturação nuclear e de antioxidantes sobre a maturação oocitária, fecundação e desenvolvimento embrionário bovino in vitro

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeito do estresse subletal com a utilização de LED no desenvolvimento e na qualidade de embriões bovinos produzidos in vitro

Pós-graduação em Medicina Veterinária - FMVZ

Biotecnologias aplicadas à reprodução de éguas velhas

The acceptance of biotechnology for the most equine breeders association had a significant effect in the horse industry, gaining popularity around the world, because the increasing on the genetic gain, allowing the use of sub fertile mares and stallions with high genetics value on reproduction. The embryos in vitro production of human and cattle has been used with success, however in vitro embryo production is not efficient in the horse, as oocyte transfer (OT) and intracytoplasmatic sperm injection (ICSI). The oocyte transfer has been used especially in subfertile old mares presenting reproductive pathologies as: endometrite, cervical and uterine adhesions, blocked oviduct, perineal laceration and ovulation failures. During oocyte recovery process, the oocytes must be collected from immature follicles that need be matured in vitro or in vivo matured oocytes from pre-ovulatory follicles through the transvaginal aspiration guided by ultrasound. The recovered oocyte is transferred to a previously inseminated recipient mare, through the flank laparotomy. The intracytoplasmatic sperm injection (ICSI) is a procedure of in vitro fertilization that needs only one sperm that is aspirated and injected inside the oocyte. The oocytes used, can be from mature and immature follicles. Fresh, cooled and frozen semen can be used, because the procedure not requires a functional sperm. The use of Piezo drill resulted in a breakthrough the pellucid zone, allowing the vibration per minute provided in the sperm injection pipette, a major result of cleaved oocytes, due to a better sperm injection in the oocyte. The embryo transfer can be straight inside the oviduct, as also transcervical transferred after embryo culture produced in vitro. In conclusion both procedures (OT and ICSI) are effective to be used on equine assisted reproduction, getting results even lower than expected, but satisfactory from animal genetically superior

A substituição do meio TCM pelo meio BARC, suplementado com SFB, BSA ou PVA, para maturação de oócitos bovinos in vitro, não incrementa a subsequente produção de blastocistos

This study was carried out to assess the influence of bovine embryo culture medium Beltsville Agriculture Research Center (BARC), supplemented with FCS, BSA or PVA, on the in vitro oocyte maturation, evidenced by cleavage rate and blastocysts production at different developmental stages. Three experiments were performed, as follows: exp.1: addition of FCS to BARC medium at concentrations of 0, 5 and 10%; exp. 2: addition of BSA to BARC medium at concentrations of 0, 4 and 8 mg/ml; exp. 3: addition of PVA to BARC medium at concentrations of 0, 0.5 and 1.0 mg/ml. TCM 199 supplemented with bicarbonate, pyruvate, gentamicin sulfate, FSH, LH and FCS was used as control group. Oocytes obtained from cow ovaries at slaughterhouse were selected in PBS, and then matured in BARC medium supplemented with FSH, LH and gentamicin sulfate, according to the experimental design. Percoll gradient was used for sperm selection and TALP medium for IVF. In vitro embryo culture was in SOF-m medium; a humidified atmosphere with 5% CO2, in air, at 38.7oC was used for all steps. The number of oocytes reaching blastocyst, expanded blastocyst, and hatched blastocyt stages was recorded, respectively at 72 and 168 h post-insemination. ANOVA and Bonferroni t test were used to determine differences among groups. Differences of P<0.05 were taken as significant. Higher percentage (P<0.05) of cleaved oocytes was observed in group TCM + FCS than for the other groups matured in BARC supplemented with FCS or BSA, regardless the concentration used. However, the cleavage rate was similar between groups BARC plus PVA with 1 mg/ml (85.7%) and TCM + FCS (90.8%). Significant difference was found among groups for the production of blastocysts, with the control group yielding a higher number of blastocysts (results ranging from 47.4 to 51.4%, in comparison with groups using BARC + FCS (4.1 to 19.7%), BSA (1.4 to 5.6%) and PVA (5.7 to 10.6%). In conclusion, BARC medium supplemented with different macromolecules did not promote a beneficial effect on in vitro oocyte maturation, resulting in lower rate of cleavage and blastocyst production when compared with TCM + FCS medium.

Efeitos de reguladores do metabolismo lipídico no desenvolvimento in vitro de embriões bovinos e na sobrevivência à vitrificação

Pós-graduação em Medicina Veterinária - FCAV

In Vitro Development and Cell Allocation After Aggregation of Syngeneic Wild Type and Fluorescence-Expressing Bovine Cloned Embryos

Background: The in vitro production (IVP) of embryos by in vitro fertilization or cloning procedures has been known to cause epigenetic changes in the conceptus that in turn are associated with abnormalities in pre- and postnatal development. Handmade cloning (HMC) procedures and the culture of zona-free embryos in individual microwells provide excellent tools for studies in developmental biology, since embryo development and cell allocation patterns can be evaluated under a wide range of embryo reconstruction arrangements and in in vitro embryo culture conditions. As disturbances in embryonic cell allocation after in vitro embryo manipulations and unusual in vivo conditions during the first third of pregnancy appear to be associated with large offspring, embryo aggregation procedures may allow a compensation for epigenetic defects between aggregated embryos or even may influence more favorable cell allocation in embryonic lineages, favoring subsequent development. Thus, the aim of this study was to evaluate in vitro embryo developmental potential and the pattern of cell allocation in blastocysts developed after the aggregation of handmade cloned embryos produced using syngeneic wild type and/or transgenic somatic cells. Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then, two enucleated hemi-oocytes were paired and fused with either a wild type (WT) or a GFP-expressing (GFP) fetal skin cell at the 11th and 19th passages, respectively. Following chemical activation, reconstructed cloned embryos and zona-free parthenote embryos were in vitro-cultured in microwells, for 7 days, either individually (1 x 100%) or after the aggregation of two structures (2 x 100%) per microwell, as follows: (G1) one WT cloned embryo; (G2) two aggregated WT embryos; (G3) one GFP cloned embryo; (G4) two aggregated GFP embryos; (G5) aggregation of a WT embryo and a GFP embryo; (G6) one parthenote embryo; or (G7) two aggregated parthenote embryos. Fusion (clones), cleavage (Day 2), and blastocyst (Day 7) rates, and embryonic cell allocation were compared by the. 2 or Fisher tests. Total cell number (TCN) in blastocysts was analyzed by the Student's test (P < 0.05). Fusion and cleavage rates, and cell allocation were similar between groups. On a per WOW basis, development to the blastocyst stage was similar between groups, except for lower rates of development seen in G3. However, when based on number of embryos per group (one or two), blastocyst development was higher in G1 than all other groups, which were similar between one another. Cloned GFP embryos had lower in vitro development to the blastocyst stage than WT embryos, which had more TCN than parthenote or aggregated chimeric WT/GFP embryos. Aggregated GFP embryos had fewer cells than the other embryo groups. Discussion: The in vitro development of GFP cloned embryos was lower than WT embryos, with no effects on cell allocation in resulting blastocysts. Differences in blastocyst rate between groups were likely due to lower GFP-expressing cell viability, as GFP donor cells were at high population cell doublings when used for cloning. On a per embryo basis, embryo aggregation on Day 1 resulted in blastocyst development similar to non-aggregated embryos on Day 7, with no differences in cell proportion between groups. The use of GFP-expressing cells was proven a promising strategy for the study of cell allocation during embryo development, which may assist in the elucidation of mechanisms of abnormalities after in vitro embryo manipulations, leading to the development of improved protocols for the in vitro production (IVP) of bovine embryos.

The role of insulin-like growth factor II and its receptor in mouse preimplantation development

Insulin-like growth factor II (IGF-II) and its receptor, the IGF-II/mannose-6-phosphate (IGF-II/M6P) receptor, are first expressed from the zygotic genome at the two-cell stage of mouse development. However, their role is not clearly defined. Insulin-like growth factor II is believed to mediate growth through the heterologous type 1 IGF and insulin receptors, whereas the IGF-II/M6P receptor is believed to act as a negative regulator of somatic growth by limiting the availability of excess levels of IGF-II. These studies demonstrate that IGF-II does have a role in growth regulation in the early embryo through the IGF-II/M6P receptor. Insulin-like growth factor II stimulated cleavage rate in two-cell embryos in vitro. Moreover, this receptor is required for the glycaemic response of two-cell embryos to IGF-II and for normal progression of early embryos to the blastocyst stage. Improved development of embryos in crowded culture supports the concept of an endogenous embryonic paracrine activity that enhances cell proliferation. These responses indicate that the IGF-II/M6P receptor is functional and likely to participate in such a regulatory circuit. The functional role of IGF-II and its receptor is discussed with reference to regulation of early development.

Oxygen-regulated gene expression in bovine blastocysts

Oxygen concentrations used during in vitro embryo culture can influence embryo development, cell numbers, and gene expression. Here we propose that the preimplantation bovine embryo possesses a molecular mechanism for the detection of, and response to, oxygen, mediated by a family of basic helix-loop-helix transcription factors, the hypoxia-inducible factors (HIFs). Day 5 compacting bovine embryos were cultured under different oxygen tensions (2%, 7%, 20%) and the effect on the expression of oxygen-regulated genes, development, and cell number allocation and HIFalpha protein localization were examined. Bovine in vitro-produced embryos responded to variations in oxygen concentration by altering gene expression. GLUT1 expression was higher following 2% oxygen culture compared with 7% and 20% cultured blastocysts. HIF mRNA expression (HIF1alpha, HIF2alpha) was unaltered by oxygen concentration. HIF2alpha protein was predominantly localized to the nucleus of blastocysts. In contrast, HIF1alpha protein was undetectable at any oxygen concentration or in the presence of the HIF protein stabilizer desferrioxamine (DFO), despite being detectable in cumulus cells following normal maturation conditions, acute anoxic culture, or in the presence of DFO. Oxygen concentration also significantly altered inner cell mass cell proportions at the blastocyst stage. These results suggest that oxygen can influence gene expression in the bovine embryo during postcompaction development and that these effects may be mediated by HIF2alpha.

Blastocyst environment and its influence on offspring cardiovascular health:the heart of the matter

The development of adult-onset diseases such as type II diabetes, obesity and cardiovascular disease is traditionally attributed to adult lifestyle characteristics such as a lack of physical exercise, poor diet and smoking. However, evidence from both human and animal model studies has demonstrated that environmental factors such as an imbalance or reduction in maternal nutrition during gestation can have adverse effects on offspring metabolism and cardiovascular health. The severity and nature of the phenotypic changes induced in offspring is influenced by the period of gestation manipulated. In particular, the mammalian preimplantation embryo in different animal models displays particular sensitivity to environmental factors, either in vivo (maternal diet) or in vitro (embryo culture) that is associated with the onset of cardiovascular dysfunction in adult life. The detailed mechanisms by which environmental conditions can alter postnatal cardiovascular physiology are poorly understood. However, various factors including endothelial function, vascular responsiveness, the renin-angiotensin system, kidney structure and early postnatal growth dynamics have all been recognize as potential contributors. Here, we review the relationship between preimplantation embryo environment and postnatal cardiovascular disease risk, and consider biochemical, molecular, genetic and physiological pathways implicated in this association. © 2009 The Authors Journal compilation © 2009 Anatomical Society of Great Britain and Ireland.

Maturation of rhesus monkey oocytes in chemically defined culture media and their functional assessment by IVF and embryo development

This study compared success of in-vitro maturation of rhesus monkey oocytes in protein-free versus serum-containing culture systems, assessed by embryo development subsequent to IVF. Four media were tested: (i) modified Connaught Medical Research Laborato

Effect of insulin-like growth factor-I during preantral follicular culture on steroidogenesis, in vitro oocyte maturation, and embryo development in mice

Insulin-like growth factor-I (IGF-I) is involved in the regulation of ovarian follicular development and has been shown to potentiate the FSH responsiveness of granulosa cells from preantral follicles. The aim of the present study was to investigate the effect of IGF-I during preantral follicular culture on steroidogenesis, subsequent oocyte maturation, fertilization, and embryo development in mice. Preantral follicles were isolated mechanically and cultured for 12 days in a simplified culture medium supplemented with 1% fetal calf serum, recombinant human FSH, transferrin, and selenium. In these conditions, follicles were able to grow and produce oocytes that could be matured and fertilized. The first experiment analyzed the effect of different concentrations of IGF-I (0, 10, 50, or 100 ng/ml) added to the culture medium on the follicular survival, steroidogenesis, and the oocyte maturation process. The presence of IGF-I during follicular growth increased the secretion of estradiol but had no effect on the subsequent oocyte survival and maturation rates. In the second experiment, IGF-I (0 or 50 ng/ml) was added to the culture medium during follicular growth, oocyte maturation, or both, and subsequent oocyte fertilization and embryo development rates were evaluated. Oocyte fertilization rates were comparable in the presence or absence of IGF-I. However, the blastocyst development rate was enhanced after follicular culture in the presence of IGF-I. Moreover, the total cell number of the blastocysts observed after differential labeling staining was also higher when follicles were cultured or matured in the presence of IGF-I.