Marco Roberto Bourg Mello

Cryopreservation of bos taurus vs bos indicus embryos: are they really different?

Cryopreservation with storage at very low temperatures is essential to make full use of this technology for both biological and commercial reasons. However, most mammalian cells will die if exposed to these temperatures unless they are exposed to cryoprotectant solutions and cooled and warmed at specific rates. Lowering temperature below 0 degree C introduces the risk of intracellular ice formation, which likely increases rapidly as the temperature falls. Evidence indicates that ice formation during cooling can cause significantly more damage than ice formation during warming. Comparisons of the toxicity of various cryoprotectants indicated that ethylene glycol (EG) is a nontoxic compound for murine and bovine embryos. The 3.6 M EG solution resulted in similar high survival rates when compared with nonfrozen embryos; deleterious effects of high concentrations of EG became apparent at 7.2 M. The use of EG provides a nontoxic method for the rapid and simplified controlled freezing of in vivo bovine compact morulae-early blastocyst, avoiding the risk of injury caused by high concentrations of cryoprotectants usually required for vitrification. However, in vivo embryos used for freezing and thawing require further studies to understand the ultrastructural changes during the freezing procedure with EG as the single cryoprotectant, especially between Holstein and Nelore cows. This paper describes the ultrastructure of bovine compact morulae-early blastocysts derived by in vivo methods from Holstein and Nelore cows to investigate the fresh morphology as well as that after exposure to cryoprotectant and cryopreservation by conventional slow freezing, quick freezing (nitrogen vapor), and vitrification.

Establishment of new murine embryonic stem cell lines for the generation of mouse models of human genetic diseases

Embryonic stem cells are totipotent cells derived from the inner cell mass of blastocysts. Recently, the development of appropriate culture conditions for the differentiation of these cells into specific cell types has permitted their use as potential therapeutic agents for several diseases. In addition, manipulation of their genome in vitro allows the creation of animal models of human genetic diseases and for the study of gene function in vivo. We report the establishment of new lines of murine embryonic stem cells from preimplantation stage embryos of 129/Sv mice. Most of these cells had a normal karyotype and an XY sex chromosome composition. The pluripotent properties of the cell lines obtained were analyzed on the basis of their alkaline phosphatase activity and their capacity to form complex embryoid bodies with rhythmically contracting cardiomyocytes. Two lines, USP-1 and USP-3, with the best in vitro characteristics of pluripotency were used in chimera-generating experiments. The capacity to contribute to the germ line was demonstrated by the USP-1 cell line. This cell line is currently being used to generate mouse models of human diseases.

A New Mouse Model for Marfan Syndrome Presents Phenotypic Variability Associated with the Genetic Background and Overall Levels of Fbn1 Expression

Marfan syndrome is an autosomal dominant disease of connective tissue caused by mutations in the fibrillin-1 encoding gene FBN1. Patients present cardiovascular, ocular and skeletal manifestations, and although being fully penetrant, MFS is characterized by a wide clinical variability both within and between families. Here we describe a new mouse model of MFS that recapitulates the clinical heterogeneity of the syndrome in humans. Heterozygotes for the mutant Fbn1 allele mgΔloxPneo, carrying the same internal deletion of exons 19–24 as the mgΔ mouse model, present defective microfibrillar deposition, emphysema, deterioration of aortic wall and kyphosis. However, the onset of a clinical phenotypes is earlier in the 129/Sv than in C57BL/6 background, indicating the existence of genetic modifiers of MFS between these two mouse strains. In addition, we characterized a wide clinical variability within the 129/Sv congenic heterozygotes, suggesting involvement of epigenetic factors in disease severity. Finally, we show a strong negative correlation between overall levels of Fbn1 expression and the severity of the phenotypes, corroborating the suggested protective role of normal fibrillin-1 in MFS pathogenesis, and supporting the development of therapies based on increasing Fbn1 expression.

Production of a cloned calf from a fetal fibroblast cell line

The present study examined the in vitro and in vivo development of bovine nuclear-transferred embryos. A bovine fetal fibroblast culture was established and used as nucleus donor. Slaughterhouse oocytes were matured in vitro for 18 h before enucleation. Enucleated oocytes were fused with fetal fibroblasts with an electric stimulus and treated with cytochalasin D and cycloheximide for 1 h followed by cycloheximide alone for 4 h. Reconstructed embryos were cultured for 7-9 days and those which developed to blastocysts were transferred to recipient cows. Of 191 enucleated oocytes, 83 (43.5%) were successfully fused and 24 (28.9%) developed to blastocysts. Eighteen freshly cloned blastocysts were transferred to 14 recipients, 5 (27.8%) of which were pregnant on day 35 and 3 (16.7%) on day 90. Of the three cows that reached the third trimester, one recipient died of hydrallantois 2 months before term, one aborted fetus was recovered at 8 months of gestation, and one delivered by cesarian section a healthy cloned calf. Today, the cloned calf is 15 months old and presents normal body development (378 kg) and sexual behavior (libido and semen characteristics).

Cryopreservation of mouse morulae through different methods: slow-freezing, vitrification and quick-freezing

The in vitro and in vivo development of mouse morulae after cryopreservation through different methods was examined. The slow-freezing involved an equilibration in 1.5M ethylene glycol (EG) and cooled at 0.5; 0.7; 1.0 or 1.2oC/minute. The vitrification involved a 3 minutes equilibration in 20% EG and 60 seconds in solution containing 40% EG, 18% ficoll and 10.26% sucrose. The quick-freezing involved an equilibration in 3M EG + 0.3M sucrose for 5 minutes and 2 minutes in nitrogen vapor. In all three methods the straws were thawed in air for 10 seconds and in water at 25oC for 20 seconds and the embryos cultured in vitro for 72 hours to estimate blastocyst rate. To assess viability in vivo, frozen morulae as well as fresh embryos were transferred into recipients. The in vitro development rates with 0.5, 0.7; 1.0 and 1.2oC/minute were, respectively, 72.3; 79.6; 76.5 and 84.8%. There was no significant difference among the cooling rates of 0.7; 1.0 and 1.2oC/minute (p > 0.01). The in vitro survival rates of vitrification and quick-freezing (84.5 and 74.3%, respectively) were similar to the slow-freezing. In vivo, the implantation rate and number of fetuses from embryos frozen through slow-freezing at 1.2oC/minute, vitrification and quick-freezing were not significantly different.

Capacitaçäo espermática in vitro com heparina e cálcio ionóforo e sua correlaçäo com a fertilidade em touros

The aim of this particular study was to test in vitro sperm capacitation protocols, using heparin (100mg/ml) and calcium ionophore (5mM). Propidium iodide and carboxifluorescein diacetate (IP/CFDA) in a fluorescence microscope as well as triple stain (congo red, neutral red and Giemsa) in Phase contrast microscope were used as staining. The spermatozoa were classified according to its viability (alive or dead) and acrossome integrity (damaged or intact). They were considered as follows: capacitated (alive and damaged); non capacitated (alive and not damaged) and dead (damaged or intact). The heparin group showed a ratio of 64.54% and 39.16% of capacitated spermatozoa in IP/CFDA and triple stain, respectively. In the calcium group, 36.41% and 18.11% of spermatozoa were capacitated in IP/CFDA and triple stain, respectively. Bulls were divided into 3 groups according to their fertility rates as follows: Group A 68%. For all three groups there was significant differences (p 0.01), when observed capacitated, non capacitated and dead spermatozoa among groups A and B; A and C; B and C, using heparin and calcium ionophore in both stains. No correlation was seen between capacitation and fertility rates. Therefore heparin treatment showed better sperm capacitation rates than calcium ionophore. The IP/CFDA technique showed itself as being a better method to evaluate sperm capacitation than the triple stain (p<0.01).