Effects of gaseous atmosphere and antioxidants on the development and cryotolerance of bovine embryos at different periods of in vitro culture

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

Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos

The aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7-9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2 degrees C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen-thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows (R). Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 +/- 5.94% and 9.43 +/- 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 +/- 3.37 and 8.67 +/- 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2 degrees C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.

Effect of melatonin on DNA damage of bovine cumulus cells during in vitro maturation (IVM) and on in vitro embryo development

The effect of melatonin during in vitro maturation (IVM) on DNA damage of cumulus cells (CCs) from bovine cumulus-oocyte complexes (COCs) and embryo development was evaluated. COCs from abattoir ovaries were cultured in maturation medium (MM) with 0.5 mu g/ml FSH and 5.0 mu g/ml LH (FSH-LH); 10(-9) M melatonin (MEL) or FSH-LH + MEL (FSH-LH-MEL). After 24 h of in vitro maturation, the CCs surrounding the oocyte were subjected to DNA analysis by Comet assay. After in vitro fertilization and in vitro embryo culture, the embryo development rates were evaluated on day 2 post insemination (cleavage) and days 7-8 (blastocyst). The percentage of CCs with no DNA damage was significantly superior in MEL group (37.6 +/- 2.4) than in FSH-LH-MEL (28.0 +/- 2.4) and FSH-LH (17.8 +/- 2.41) groups. Cleavage and blastocysts rates were similar among groups. Melatonin during IVM protects the CCs from DNA damage but this effect did not influence embryo development in vitro. (C) 2010 Elsevier Ltd. All rights reserved.

Effect of antibiotics on development in vitro of hamster pronucleate ova

Antibiotics are commonly added to embryo culture media, but effects on embryo development have not been examined thoroughly. Hamster ova were used to investigate whether penicillin, streptomycin or gentamicin affect embryo development in vitro. Ova were collected 10 h post activation by spermatozoa in vivo and cultured in five treatments: 1) Control: chemically-defined medium HECM-9 with no antibiotics; 2) HECM-9 with 100 IU/mL, penicillin; 3) HECM-9 with 50 mug/mL streptomycin; 4) HECM-9 with 10 mug/mL,gentamicin and 5) HECM-9 with both 100 IU/mL penicillin and 50 mug/mL streptomycin. Individually, penicillin, streptomycin and gentamicin did not affect embryo development to the 8-cell stage at 58 h post oocyte activation, or morula/blastocyst stages, or blastocysts alone at 82 h post activation. However, when penicillin and streptomycin were both present in the culture medium the percentages of 8-cell embryos at 58 h and blastocysts at 82 h were significantly lower than the control. No antibiotic treatment improved hamster embryo development in vitro. We caution against the use of penicillin and streptomycin together for hamster embryo culture, and show that it is not necessary to include any antibiotics in embryo culture media for up to 72 h if proper sterile technique is used with an oil overlay. (C) 2000 by Elsevier Science Inc.

Messenger RNA expression of Pabpnl1 and Mbd3l2 genes in oocytes and cleavage embryos

Objective: To identify genes specifically expressed in mammalian oocytes using an in silico subtraction, and to characterize the mRNA patterns of selected genes in oocytes, embryos, and adult tissues. Design: Comparison between oocyte groups and between early embryo stages. Setting: Laboratories of embryo manipulation and molecular biology from Departamento de Genetica (FMRP) and Departamento de Ciencias Basicas (FZEA) - University of Sao Paulo. Sample(s): Oocytes were collected from slaughtered cows for measurements, in vitro fertilization, and in vitro embryo culture. Somatic tissue, excluding gonad and uterus tissue, was collected from male and female cattle. Main Outcome Measure(s): Messenger RNA levels of poly(A)-binding protein nuclear-like 1 (Pabpnl1) and methyl-CpG-binding domain protein 3-like 2 (Mbd3l2). Result(s): Pabpnl1 mRNA was found to be expressed in oocytes, and Mbd3l2 transcripts were present in embryos. Quantification of Pabpnl1 transcripts showed no difference in levels between good-and bad-quality oocytes before in vitro maturation (IVM) or between good-quality oocytes before and after IVM. However, Pabpnl1 transcripts were not detected in bad-quality oocytes after IVM. Transcripts of the Mbd3l2 gene were found in 4-cell, 8-cell, and morula-stage embryos, with the highest level observed in 8-cell embryos. Conclusion(s): Pabpnl1 gene expression is restricted to oocytes and Mbd3l2 to embryos. Different Pabpnl1 mRNA levels in oocytes of varying viability suggest an important role in fertility involving the oocyte potential for embryo development. (Fertil Steril (R) 2010; 93: 2507-12. (C) 2010 by American Society for Reproductive Medicine.)

In Vitro Propagation of Heliconia bihai (L.) L. from Zygotic Embryos

(In vitro Propagation of Heliconia bihai L. from Zygotic Embryos). The internal morphology of embryos from immature and mature fruits of Hcliconia bihai (L.) L. cv. Lobster Claw Two was examined. Embryos were inoculated into MS media (full MS and 1/2 MS) and GA(1) (0.2.5 and 5 mg L(-1)) with either sucrose or glucose. These plantlets were then replicated and transferred to MS medium (full MS or 1/2 MS) with 0 or 2.5 mg L(-1) BAP and their multiplication was evaluated 30 and 45 days after inoculation. The genetic variability of the multiplied plants was estimated using isoenzyme analyses. The internal morphology of the mature embryos revealed their tissues to be in more advanced stages of differentiation than immature embryos. In the conversion phase, 85% of the inoculated embryos developed into plants in the 1/2 MS medium with sucrose, in contrast to only 41% of the embryos that were cultivated with glucose. In the multiplication phase, plants cultivated in 1/2 MS medium with 2.5 mg L(-1) BAP demonstrated more buds. Isoenzyme analyses showed pattern changes in terms of the color intensity and the migration of some of the bands. These results may be associated with differences in the ages of the mother plants and of the plantlets obtained in vitro.

Identificação de híbridos de citros resistentes à mancha-marrom-de-alternária por meio de fAFLP e testes de patogenicidade

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

Development of globular embryos from the hybridization between 'Pêra Rio' sweet orange and 'Poncã' mandarin

Objetivou-se estudar a influência de diversas concentrações do meio MT, sacarose, vitaminas, carvão ativado e ácido giberélico no cultivo de embriões imaturos oriundos do cruzamento entre laranjeira 'Pêra Rio' x tangerineira 'PONCÃ' . Os embriões foram excisados sob condições assépticas e inoculados em 15 mL do meio de cultura MT, de acordo com cada experimento a seguir: 1) concentrações do meio de cultura MT (0%, 50%, 100%, 150% e 200%) combinados com 0, 30, 60 e 90 g.L-1 de sacarose; 2) concentrações de vitaminas do meio MT (0%, 50%, 100%, 150% e 200%) combinados com 0, 30, 60 e 90 g.L-1 de sacarose e; 3) concentrações de carvão ativado (0; 0.5; 1; 1.5 e 2 g.L-1) combinados com GA3 (0; 0.01; 0.1; 1 e 10 mg.L-1). Após a inoculação, os embriões foram mantidos por 90 dias em sala de crescimento à temperatura de 27±1ºC, fotoperíodo de 16 horas e irradiância de 32 µmol.m-2.s-1. A utilização de 50% e 100% do meio MT associado a 60 e 90 g.L-1 de sacarose, respectivamente, acrescido de 0.01 mg.L-1 de GA3, proporcionou melhor desenvolvimento de embriões globulares. Não houve necessidade da adição de carvão ativado e vitaminas no meio MT para o cultivo de embriões globulares.

Cultura de embrião e indução de brotos in vitro para micropropagação do pinhão-manso

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

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)

Fetotoxicity caused by the interaction between zinc and arsenic in mice

Arsenic is an environmental pollutant that induces congenital malformations in experimental models and can contribute to human birth defects. The environmental exposure to arsenic is relatively small when compared with the doses required to cause teratogenicity in mice and other laboratory animals. In order to study the action of zinc in the arsenic-induced teratogenicity, in the present work mice were either pretreated with zinc and later with arsenic or were treated simultaneously with zinc and arsenic in vivo and in vitro. Following administration of arsenate on gestation day 8, pregnant females were killed on the 17th day of gestation; maternal and fetal data were collected by laparotomy and used to calculate reproductive parameters. Fetuses were analyzed for the presence of external malformation and, after the appropriate processing, visceral and skeletal analyses were accomplished. Conceptuses were exposed in whole embryo culture to arsenicals on gestation day 8 (3-6 somite stage). After a 26 h culture period, morphological development was assessed. Neither pretreatment with zinc nor simultaneous administration of zinc prevented arsenic teratogenicity in these experimental models. (C) 2002 Wiley-Liss, Inc.

Evaluation of serum- and animal protein-free media for the production of infectious bronchitis virus (M41) strain in a continuous cell line

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Embryos refrozen-thawed by vitrification lead to live births: Case report

Objective: To describe two successful cases of utilizing refrozen blastocysts by vitrification derived from supernumerary embryos. Design: Case report. Setting: Private fertility clinic. Subjects: Two infertility couple. Interventions: Refrozen blastocysts by vitrification derived from supernumerary embryos. Main outcome measures: Obstetric and pediatric results. Results: Two pregnancies obtained from refrozen-warmed blastocysts led to two healthy babies being born without clinical or genetic problems. Conclusion: These case reports support the notion of safely repeating cryopreservation. However, despite these favorable results, there is still a need for prospective controlled studies on the obstetric and neonatal repercussions of refreezing and of vitrification in particular. © 2010 Middle East Fertility Society.

Valor preditivo do potencial de implantação embrionário pela análise do perfil químico de meios de cultivo por espectrometria de massas (ESI-Q-ToF)

Objective: This case-control study analyzed mass spectrometry fingerprinting patterns of culture media samples used for embryo culture to predict embryo implantation. Methods: The culture medium harvested after embryo transfer of 22 embryos from 13 patients was used for the experiments. After embryo transfer, the remaining culture media were collected and samples were split in positive (n=8) and negative (n=14) implantation groups according to implantation outcomes (100% or 0% of implantation). Samples were individually diluted and injected directly to the Electrospray ionization (ESI) MS coupled to a Quadrupole Time-of-flight MS (Q-ToF-MS).Ions relative intensities of each spectrum were considered. Data analysis was conducted in MatLab 7.0 version using Partial Least Squares - Discriminant Analysis toolbox. Results: There were 3027 observed ions at 100% and 0% implantation groups by ESI-Q-ToF-MS. The statistical model could categorize the samples in two clusters, based on their positive and negative implantation outcomes. Less intense ions present in the mass spectra with statistical significance have contributed to the major differences to group distinction. Conclusions: Positive and negative implantation embryos showed a specific biochemical pattern present in culture media, which could be detected as a fast, simple and non-invasive way. This biochemical profile could help the selection of the most viable embryo, improving single embryo transfer and thus eliminating the risk and undesirable outcomes of multiple pregnancies. © Todos os direitos reservados a SBRA - Sociedade Brasileira de Reprodução Assistida.

Revisando os efeitos da concentração de oxigênio sobre o desenvolvimento embrionário

Despite the fact that the processes of embryo development and implantation of mammals do not occur in atmospheric tension in vivo, it is common practice in laboratories for in vitro fertilization (IVF) the culture of embryos in an oxygen tension of 20%. The discrepancy between the tensions of oxygen leads to important discussions about their effects in vitro embryo development. It is believed that the culture of embryo in atmospheric oxygen tension can be detrimental to the quality of the data for predisposing them to the negative interference of reactive oxygen species (ROS), resulting in oxidative stress, leading to deleterious effects on metabolism embryo gene expression and blastocysts grown in those conditions. Faced with arguments for and against the culture of embryos in vitro oxygen tensions similar to those found in vivo, this revies article aims to condense and discuss the literature concerning the influence of oxygen tension on the quality of in vitro culture.