The effect of interaction between macromolecule supplement and oxygen tension on bovine oocytes and embryos cultured in vitro

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

Effects of fetuin on zona pellucida hardening, fertilization and embryo development in cattle

Mammalian oocytes can undergo spontaneous meiotic maturation when they are liberated from their follicles and cultured in vitro; however, the zona pellucida (ZP) becomes resistant to chymotrypsin digestion, or hardens, when spontaneous maturation occurs in serum-free medium. Schroeder et al. [Biol. Reprod. 43 (1990) 891] described that fetuin, a component of fetal calf serum (FCS), inhibits ZP hardening during oocyte maturation. The aim of this experiment was to study the effect of the presence of cumulus cells and addition of hormones to maturation media on bovine zona hardening and embryo development in medium with and without fetuin. In Experiment 1, different concentrations of fetuin were added to the maturation medium. The time necessary for digestion of 50% of the ZP (d50) was not different when oocytes were matured in presence of 10% FCS, 1 mg/ml polyvinyl alcohol (PVA), or 4, 1 and 0.25 mg/ml of fetuin; cleavage rates were also similar. However, significantly more blastocysts (P < 0.05) were formed when FCS was used compared to PVA and 0.25 mg/ml of fetuin. In Experiment 11, we examined the influence of the presence of cumulus cells and hormones during the maturation of oocytes in media with PVA, BSA, FCS and fetuin. The d50 was significantly higher (P < 0.05) when oocytes were matured in presence of cumulus cells. The cleavage rate of cumulus-intact oocytes was similar for all groups. However, when oocytes were partially stripped before maturation, the cleavage rate was significantly higher (P < 0.05) when FCS or fetuin was used. In both stripped and non-stripped groups, significantly more blastocysts (P < 0.05) were formed when oocytes were matured with FCS compared to BSA and PVA. These results indicate that zona hardening, as described for mouse and human oocytes, does not have a large effect on bovine cumulus-intact oocytes. Apparently fetuin can be used as a substitute for FCS during bovine oocyte maturation, since it leads to similar developmental rates as FCS in intact and partially stripped oocytes. (C) 2002 Published by Elsevier B.V. B.V.

Lipid content and apoptosis of in vitro-produced bovine embryos as determinants of susceptibility to vitrification

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

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)

THE EFFECT OF SERUM ON IN-VITRO MATURATION, IN-VITRO FERTILIZATION AND STEROIDOGENESIS OF BOVINE OOCYTES COCULTURED WITH GRANULOSA-CELLS

The influence of fetal calf serum alone (FCS) or associated with proestrous (FCS+PCS), estrous (FCS+ECS) or metaestrous (FCS+MCS) cow serum added to the culture medium and of the steroids produced by co-cultured granulosa cells were evaluated in terms of the in vitro maturation (TVM) and fertilization (IVF) of bovine oocytes. Supplementation of the medium with FCS+ECS and FCS+MCS resulted in higher proportions of oocytes that reached metaphase II (96.0% and 93.3%, respectively) and in higher proportions of embryos that reached the four- and eight-cell/morula stages (51.9% and 65.6%, respectively), whereas the supplementation with FCS and FCS+PCS resulted in only 79.2% and 67.5%, respectively, of matured oocytes and 26.7% and 34.3%, respectively, of cleaved embryos. These findings show that the best IVM and IVF were obtained at lower concentrations of estradiol produced by co-cultured granulosa cells (supplementation with FCS+ECS: 10.3 ng/ml and FCS+MCS: 2.1 ng/ml), whereas the worst-results in IVM and IVF occurred at higher concentrations of estradiol that were obtained with FCS (33.1 ng/ml) and FCS+PCS (19.9 ng/ml) supplementation. These data suggest an inhibitory effect of estradiol on resumption of oocyte meiosis in vitro.

The effect of serum on in vitro maturation, in vitro fertilization and steroidogenesis of bovine oocytes co-cultured with granulosa cells.

The influence of fetal calf serum alone (FCS) or associated with proestrous (FCS+PCS), estrous (FCS+ECS) or metaestrous (FCS+MCS) cow serum added to the culture medium and of the steroids produced by co-cultured granulosa cells were evaluated in terms of the in vitro maturation (IVM) and fertilization (IVF) of bovine oocytes. Supplementation of the medium with FCS+ECS and FCS+MCS resulted in higher proportions of oocytes that reached metaphase II (96.0% and 93.3%, respectively) and in higher proportions of embryos that reached the four- and eight-cell/morula stages (51.9% and 65.6%, respectively), whereas the supplementation with FCS and FCS+PCS resulted in only 79.2% and 67.5%, respectively, of matured oocytes and 26.7% and 34.3%, respectively, of cleaved embryos. These findings show that the best IVM and IVF were obtained at lower concentrations of estradiol produced by co-cultured granulosa cells (supplementation with FCS+ECS: 10.3 ng/ml and FCS+MCS: 2.1 ng/ml), whereas the worst results in IVM and IVF occurred at higher concentrations of estradiol that were obtained with FCS (33.1 ng/ml) and FCS+PCS (19.9 ng/ml) supplementation. These data suggest an inhibitory effect of estradiol on resumption of oocyte meiosis in vitro.

Cryopreservation of equine embryos with glycerol plus sucrose and glycerol plus 1,2-propanediol.

Six or 7-day-old equine embryos were divided into 4 groups; Group 1, n = 15, Day 7 embryos destined for immediate transfer; Group 2, n = 15, Day 6 embryos destined for deep-freezing with glycerol plus sucrose as cryoprotectant; Group 3, n = 10, Day 6 embryos destined for deep-freezing with glycerol plus 1,2-propanediol as cryoprotectant and Group 4, n = 3, fresh embryos destined for ultrastructural analysis. All the frozen/thawed embryos were transferred to recipient mares, except 3 embryos in Group 3 that were subjected to ultrastructural analysis. After thawing the cryoprotectants were removed by successive dilutions in PBS + 15% v:v fetal calf serum (FCS) containing decreasing concentrations of the cryoprotectants. Pregnancy was diagnosed ultrasonographically in 53.3%, 13.3% and 0% of the mares in Groups 1, 2 and 3 respectively. Ultrastructural analysis showed differences between frozen/thawed and fresh embryos. In the former, embryonic cells were deformed and showed dilation of the intercellular and perivitelline spaces, a decrease of desmosome number in the junctional complexes, few microvilli on the apical surface of the trophectoderm and an almost total absence of pinocytotic vesicles. Most of the mitochondria showed regions containing dilation and irregularities on the cristae, which appeared electron-dense. The results obtained with Groups 2 and 3 embryos showed that the cryoprotectants employed were not effective in protecting the embryos against damage during freezing and thawing. Indeed, the ultrastructural changes observed in the Group 3 embryos explained the absence of any established pregnancies in this group of mares.

Efeitos de diferentes sistemas de cultivo in vitro sobre o crescimento de folículos pré-antrais isolados de ovários de fetos bovinos

The objective of this study is to use different in vitro culture systems of preantral follicles from Nelore breed bovine fetuses in the last gestation quarter. The evaluation of treatments considered the time of growth of isolated follicles. Preantral follicles were mechanically isolated and submitted to the individual culture, for 9 days, in media no supplemented or supplemented with fetal calf serum (FCS), bovine serum albumin (BSA) or synthetic defined supplement substitute of serum KnockoutSR (KNO). We have also evaluated the effects of collagen gel or fetal calf fibroblast monolayer as substratum for in vitro cultures. The increase on the follicular diameter was followed in the first day (0 h), at the 72 h, 144 h and 216 h. Considering cultures of isolated follicles, the results have shown that the association between media supplemented with FCS and collagen gel was significantly more efficient on the increase of the follicular diameter than other treatments. It is not still established a system of appropriate cultivation that sustains the differentiation and multiplication of the granular cells and that maintains the contact of the same ones with the oocyte to provide molecules and factors that supply the metabolic demand. We also understand that our results also represent another promising step on the search for the ultimate system of in vitro culture of preantral follicles from bovines.

Avaliação de gametas e embriões bovinos produzidos in vitro após exposição experimental ao BoHV-5

Pós-graduação em Ciência Animal - FMVA

Obtenção, manutenção e caracterização de células-tronco embrionárias equinas

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

Vitrificação de embriões bovinos produzidos in vivo e in vitro após lipólise química

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

Estudo morfofisiométrico de ovários e maturação ovocitária in vitro em bubalinos e bovinos nas diferentes fases da atividade reprodutiva

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Contribuição para a clonagem em eqüinos por meio de tranferência nuclear

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

Influência da adição de fator de crescimento fibroblástico 10 (FGF10), durante a maturação oocitária, na produção in vitro de embriões bovinos

The Brazilian livestock stands out for having the world largest commercial herd of cattle and leads meat exportation and production of bovine embryos. The in vitro production (IVP) of embryos is considered an effective option to overcome problems such as infertility in cows with high economic value and also for genetic improvement of cattle. The in vitro oocyte maturation is an essential step to the success of IVP, but is still considered poor when compared to in vivo maturation. Recent studies have suggeested an important role of Fibroblast Growth Factor 10 (FGF10) on the in vitro maturation of oocytes, which favored the expression of genes related to oocyte maturation and cumulus cell expansion. Aware that maturity stage influences the final production of blastocysts, we aimed study to verify if the addition of FGF10 into the maturation medium is able to affect positively the IVP of bovine embryos. Hence, FGF10 was added to maturation in five different concentrations: 0.5 ng/mL (group 0.5), 2.5 ng/mL (group 2.5), 5 ng/mL (group 5), 10 ng/mL (group 10) and 50 ng/mL (group 50). Additionally, two other maturation groups were used, group BSA (Bovine Serum Albumin, 4 mg/mL) and group FCS (Fetal Calf Serum, 10%). The rates of cleavage, morula and blastocyst were analyzed by Analysis of Variance (ANOVA), differences of P<0.05 were considered significant. Cleavage rates did not differ between the seven groups. On the other hand, morula rate on FCS group was higher than groups BSA, 0.5, 10 and 50 (P<0.05), but did not differ among groups treated with intermediate doses of FGF10 (2.5 and 5). FCS group presented higher blastocyst rate compared to all other groups that were well below the FCS group (P<0.0001). Therefore, the use of FGF10 during oocyte maturation did not affect positively embryo development on the IVP of bovine embryos

Influência da luz LED em culturas de células-tronco mesenquimais in-vitro

Stem cells are defined as cells capable of self-renewal and differentiation into specialized cells when submited to external signalings in the enviroment. Among adult stem cells, mesenchymal cells occupy an important position because they can differentiate into mesodermal cells such as osteoblasts, adipocytes and chondrocytes. Cell therapy consists in the use of mesenchymal stem cells (MSC) in the treatment of degenerative diseases and harmed tissue reconstruction. Due to the longstanding and costly procedure for cultivation of MSC, it was proposed the use of low power light sources, such as light emitting diodes (LED), to optimize these factors. Recent works have shown a series of results from the influence of LED light on biological tissues such as increased rate of cell proliferation, increased RNA, DNA and ATP synthesis rate. The purpose of this study is to compare the biomodulator effect of LED light set at wavelengths 630nm ± 10nm and 805nm ± 10nm on the mesenchymal stem cells proliferation. For this, the mesenchymal stem cells culture adopted the procedure used in the Departament of Animal Reproduction and Veterinary Radiology of the Faculty of Veterinary Medicine and Animal Sciences of Botucatu. MSC were obtained from an adult horse bone marrow, and isolated by density gradient separation, with the FICOLL reagent and by centrifugation. The pellet containing the stem cells was removed and these were placed in low glucose DMEM culture medium, containing 10% fetal calf serum and antibiotics. The material was observed daily by inverted microscopy for monitoring the progression of the cells and subsequently the amount of cells were counted in a Neubauer counting chamber. The amount of MSC was obtained by cell culture seeded in 24 wells culture plate and segregated into three distinct groups: Group 1 was irradiated with wavelength set at 630nm ± 10 nm, Group... (Complete abstract click electronic access below)