Sexing single bovine blastomeres using TSPY gene amplification.

The testis-specific protein Y-encoded gene (TSPY) is a Y-specific gene present in variable copy number in many mammalian species, including cattle. We tested the applicability of the TSPY gene as a Y-specific marker to predict preimplantation embryo sex in Nelore (Bos indicus) cattle. Two blastomeres were removed from each embryo. A total of 36 single blastomeres and the remaining cells of their 18 matched in vitro conceived embryos were screened for TSPY amplification by nested-PCR. The results obtained from a single blastomere and the remaining cells of the same embryo were concordant in all cases. All blastomeres (16/16) from eight embryos produced with sexed sperm (specific for production of male embryos) were TSPY-positive. We conclude that TSPY is a good male-specific marker, the usefulness of which is probably enhanced by the high copy number. Other methods that are less time-consuming, such as real-time PCR, could be improved with the use of the TSPY gene sequences to generate primers and/or probes. This is the first report to demonstrate the applicability of the TSPY gene for sexing single cells in cattle.

Bovine fetal DNA in the maternal circulation: Applications and implications.

OBJECTIVE: The main aim of the present study was to detect bovine fetal DNA in the maternal circulation, a relatively unexplored subject in the literature. STUDY DESIGN: DNA was extracted from blood of 84 primipara cows (Bos indicus) at different gestational ages (30-270 days) and from 100 adult animals (50 males and 50 non-pregnant cows). The samples were analyzed using PCR with primers for TSPY gene. RESULTS: Molecular results matched the fetal phenotypic gender in all 47 male and 37 female fetuses, including early pregnancy, and in control animals. CONCLUSIONS: These results evidence a bovine transplacental fetal DNA passage.

Formocresol mutagenicity following primary tooth pulp therapy: an in vivo study.

OBJECTIVES: To investigate whether formocresol, in Buckley's original formulation, is mutagenic in vivo to lymphocyte cultures obtained from the peripheral blood of children aged from 5 to 10 years old. These children were recruited from those attending the dental clinics of Recife City Council and the University of Pernambuco School of Dentistry, Brazil. METHODS: The sample comprised 20 children who had primary teeth with cariously exposed vital pulps. Two venous blood samples were collected (6-8 ml) from each child, the first prior to vital pulpotomy (control group) and the second 24 h after pulpotomy (treated group). This research is a case-control study. The peripheral lymphocytes were grown in a complete culture medium consisting of 78% RPMI 1640 medium (a), supplemented with streptomycin (0.01 mg/ml), penicillin (0.005 ml(-1)), 20% fetal bovine serum (b) and 2% phytohemagglutinin (c). The lymphocytes were assessed for chromosomal aberrations via a previously published method which was modified. The cytogenetic analysis was performed in a blind test, where the slides were codified by an annotator and the scorers did not know which group they were analyzing. For each sample, this envolved the analysis of 200 metaphases. The level of significance adopted in the statistical test was 5.0% (p<0.05). RESULTS: There was no statistically significant difference in clinical doses between the control and treated groups, using Wilcoxon's Signed Ranks test, for the chromosomal aberrations (P=0.251) and for the total chromosomal breaks (P=0.149). Although there were no statistically significant differences between the control and treated groups, Buckley's formocresol was mutagenic for one patient, raising doubt about the desirability of its use for pulpotomies in children. CONCLUSIONS: The results revealed that, from a statistical standpoint, formocresol is not mutagenic. However, further investigations are required, preferably with a larger sample, in patients needing more than one pulpotomy in order to observe whether an increase in the quantity of the drug would increase the quantity of chromosome aberrations and also to verify individual susceptibility to chromosome alterations with the use of formocresol.