Post-thaw viability of in vivo-produced canine blastocysts cryopreserved by slow freezing

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Melatonin in maturation media fails to improve oocyte maturation, embryo development rates and DNA damage of bovine embryos

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

Influência do fator de crescimento semelhante à insulina I (IGF-I) adicionado ao meio fluido sintético de tuba uterina (SOF) sobre a maturação in vitro de oócitos caninos (Canis familiaris)

The follicular growth and oocyte maturation knowledge are very important to the development and improvement of new biotechnologies such as in vitro fertilization and somatic cell nuclear transfer. In order to the necessity of clarify the basic mechanisms related to canine oocyte maturation, this investigation focuses on the evaluation of the effect of insulin-like growth factor-i (IGF-I), added to synthetic oviductal fluid medium (SOF) on the in vitro maturation of domestic dog oocytes. Thirty-seven bitches undergoing ovariohysterectomy for castration or due to pathological conditions of the uterus were selected as oocytes' donors (n=875). The oocytes were allocated in the following groups: MO (stained in the collection's time), Control (72h in SOF) and Experimental (72h in SOF plus 100 ng IGF-I). After 72 hours of maturation the oocytes' nuclear status were assessed by Hoechst 33342 dye. The best results in terms of oocyte harvest were observed in those juvenile donors,, females in estrus, nuliparous and pure breeds. No significant differences were observed between treatments control (SOF) or experimental (IGF-I).

Role of roscovitine and IBMX on kinetics of nuclear and cytoplasmic maturation of bovine oocytes in vitro

The 3-isobutyl-1-methylxanthine (IBMX) is able to prevent resumption of meiosis by maintaining elevated cyclic AMP (cAMP) concentrations in the oocyte, and roscovitine, a purine known to specifically inhibit MPF kinase activity, maintains bovine oocytes at the germinal vesicle (GV) stage. The present study was conducted to analyze whether cytoplasmic maturation (examined by the pattern of cortical granule (CG) distribution) of bovine oocytes is improved during meiotic arrest with IBMX and roscovitine. Oocytes were matured in vitro in a 10% Knockout(SR) supplemented TCM-199 medium (Control) with either 0.5 mM IBMX or 25 mu M roscovitine (ROSC). Oocytes were stained with fluorescein isothiocyanate conjugated Lens culinaris agglutinin (FITC-LCA) for CG evaluation and with Hoechst 33342 for nuclear stage assessment. At 16 h of culture, the percentage of oocytes remaining in the GV stage was higher (P < 0.05) in the ROSC group (32.41%) compared with the Control and IBMX groups (8.61% and 9.73%, respectively). At 24h of culture, progression of meiosis to M II stage was retarded (P < 0.05) in the ROSC group (24.05%) compared to the Control (60.20%), whereas the IBMX group (33.88%) showed no significant difference to the other two groups. At 16h of maturation, the proportion of oocytes with CG in clusters (immature cytoplasm) was similar between the groups, as was the percentage of peripheral CG (mature) at 24h of maturation. The results of the present study demonstrated that the meiotic inhibitors IBMX and roscovitine delay the progression of nuclear maturation without affecting cytoplasmic maturation, assessed by the analysis of CG repositioning. (c) 2006 Elsevier B.V. All rights reserved.

Evaluation of developmental changes in bovine in vitro produced embryos following exposure to bovine Herpesvirus type 5

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

Wolbachia endosymbiont in a species of the Anastrepha fraterculus complex (Diptera: Tephritidae)

In Anastrepha sp.2 aff. fraterculus, the egg-cell harbours a large population of endosymbionts. The bacteria were identified as belonging to genus Wolbachia by PCR assay using primers of the ftsZ gene followed by sequencing of the amplified band. Newly deposited eggs stained in toto by Hoechst show that the bacteria are unevenly dispersed throughout the egg-cell, with a higher accumulation at the posterior pole, and that the degree of infestation varies from egg to egg. Analysis by transmission electron microscopy shows that bacteria are present in the female germ line of embryonic and larval stages, as well as in the different cell types of the ovaries at the adult stage. Mature ova within the follicles harbour a large population of the symbionts. The results indicate the existence of a transovarian transmission of the endosymbionts in this fly.

HDAC inhibition decreases XIST expression on female IVP bovine blastocysts

During initial development, both X chromosomes are active in females, and one of them must be silenced at the appropriate time in order to dosage compensate their gene expression levels to male counterparts. Silencing involves epigenetic mechanisms, including histone deacetylation. Major X chromosome inactivation (XCI) in bovine occurs between hatching and implantation, although in vitro culture conditions might disrupt the silencing process, increasing or decreasing X-linked gene expression. In this study, we aimed to address the roles of histone deacetylase inhibition by trichostatin A (TSA) on female preimplantation development.We tested the hypothesis that by enhancing histone acetylation, TSA would increase the percentage of embryos achieving 16-cell stage, reducing percentage of embryos blocked at 8-cell stage, and interfere with XCI in IVF embryos. We noticed that after TSA treatment, acetylation levels in individual blastomeres of 8-16 cell embryos were increased twofold on treated embryos, and the samewas detected for blastocysts. Changes among blastomere levels within the same embryo were diminished on TSA group, as low-acetylated blastomeres were no longer detected. The percentage of embryos that reached the 5th cleavage cycle 118 h after IVF, analyzed by Hoechst staining, remained unaltered after TSA treatment. Then, we assessed XIST and G6PD expression in individual female bovine blastocysts by quantitative real-time PCR. Even though G6PD expression remained unaltered after TSA exposure, XIST expression was eightfold decreased, and we also detected a major decrease in the percentage of blastocysts expressing detectable XIST levels after TSA treatment. Based on these results, we conclude that HDAC is involved on XCI process in bovine embryos, and its inhibition might delay X chromosome silencing and attenuate aberrant XIST expression described for IVF embryos. © 2013 Society for Reproduction and Fertility.

Vitrification of immature feline oocytes with a commercial kit for bovine embryo vitrification

The aim of this study was to evaluate the suitability of a commercial kit for bovine embryo vitrification for cryopreserving cat oocytes and to evaluate comparatively the effects of its use with slow freezing procedure on cryotolerance in terms of morphology and oocyte resumption of meiosis. Germinal vesicle stage oocytes isolated from cat ovaries were either vitrified (n=72) using a vitrification kit for bovine embryo or slow frozen (n=69) by exposing oocyte to ethylene glycol solution before being transferred to a programmable embryo freezer. After thawing and warming, oocytes were cultured for 48h and then were examined for meiosis resumption using bisbenzimide fluorescent staining (Hoechst 33342). Fresh immature oocytes (n=92) were used as the control group. The proportion of oocytes recovered in a morphologically normal state after thawing/warming was significantly higher in frozen oocytes (94.5%) than in the vitrified ones (75%, p<0.01). Morphological integrity after culture was similar in vitrified (73.6%) and slow frozen oocytes (76.8%); however, only 37.5% of the morphologically normal oocytes resumed meiosis after vitrification compared to 60.9% of those submitted to slow freezing procedure (p<0.01). Fresh oocytes showed higher morphological integrity (91.3%) and meiosis resumption rates (82.6%, p<0.002) than cryopreserved oocytes, irrespective of the procedure used. These results suggest that immature cat oocytes vitrified with a kit for bovine embryos retain their capacity to resume meiosis after warming and culture, albeit at lower rates than slow frozen oocytes. Vitrification and slow freezing methods show similar proportions of oocytes with normal morphology after culture, which demonstrate that thawed and warmed oocytes that resist to cryodamage have the same chances to maintain their integrity after 48h of culture. © 2012 Blackwell Verlag GmbH.

Avaliação dos efeitos da exposição experimental ao herpesvírus bovino tipo 5 em embriões bovinos produzidos in vitro

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

Efeito antitumoral, genotóxico e mutagênico de nitensidina A em camundongos nude BALB/c com implante tumoral xenográfico de células imortalizadas com HPV-16 (SiHa)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Atividade citotóxica, pró-apoptótica, genotóxica e mutagênica de nitensidina B em células tumorais cervicais humanas

Pós-graduação em Biociências e Biotecnologia Aplicadas à Farmácia - FCFAR

Avaliação de apoptose induzida por extrato orgânico de material particulado atmosférico, rico em hidrocarbonetos policíclicos aromáticos, na região urbana de Araraquara durante a safra e entressafra de cana-de-açúcar

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeito do FGF10 na maturação oocitária e produção de embriões bovinos in vitro

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Produção de embriões por fecundação in vitro e ativação partenogenética visando o isolamento de células tronco embrionárias felinas

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