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Sanna D.,Laboratory of Animal Reproduction and Biotechnology | Sanna D.,University of Sassari | Sanna A.,Neuroscienze PharmaNess Scarl Edif 5 Parco Scientifico Sardegna Ricerche Loc | Mara L.,Laboratory of Animal Reproduction and Biotechnology | And 5 more authors.
Cell Biology International | Year: 2010

Transcription factor Oct4 (octamer-binding transcription factor-4) is important in early embryonic development and differentiation. It is also required for maintenance of pluripotency of the inner cell mass, and is used as a staminality marker of embryonic stem cells. Changes in Oct4 expression during the different stages of early embryo development have been reported, and therefore we have conducted a quantitative study of Oct4 gene expression of sheep blastocysts in vitro, and of embryonic-stem-like cells at the undifferentiated stage and in the course of differentiation. To characterize embryonicstem-like cells, alkaline phosphatase activity, stage-specific embryonic surface antigens SSEA-1, SSEA-3, SSEA-4 and three specific gene markers Nanog, Sox2 and Stat3 were assayed. cDNA produced by RT (reverse transcriptase)-PCR was synthesized and amplified by PCR; sequencing gave 98, 95 and 98% homology with the bovine sequences of Oct4, Nanog and Stat3 respectively. Using the ovine sequence of 290 bp, quantitative expression of Oct4 in the inner cell mass, trophoblast and embryonic-stem-like cells was performed by qRT-PCR (quantitative real-time PCR). Oct4 was expressed in the inner cell mass, trophoblast and embryonic-stem-like cells. Expression in the inner cell mass was significantly higher than in the trophoblast. This could be useful in defining the quality of embryos produced and makes it possible to use Oct4 to detect pluripotency. In addition, the different levels of Oct4 expression between undifferentiated and differentiating embryonic-stem-like cell cultures could be used to detect this gene as a staminality marker. © The Author(s) Journal compilation. © 2010 Portland Press Ltd.

Maria A.N.,Laboratory of Animal Reproduction and Biotechnology | Azevedo H.C.,Laboratory of Animal Reproduction and Biotechnology | Santos J.P.,Laboratory of Animal Reproduction and Biotechnology | Silva C.A.,Laboratory of Animal Reproduction and Biotechnology | Carneiro P.C.F.,Laboratory of Animal Reproduction and Biotechnology
Journal of Applied Ichthyology | Year: 2010

Seminal features of tambaqui were evaluated after hormonal induction of spermiation with common carp pituitary extract. Seventeen adult (6.1±0.9-kg, 62±6-cm) males were collected from earthen ponds and transported to indoor concrete tanks. Semen was evaluated according to volume, pH, osmolality, motility, concentration, viability, sperm morphometry and morphological abnormalities. The semen of tambaqui was white and milky. The volume was 10.2±5.1-mL at pH 8.0±0.1, and yielded a concentration of 9.1×109-spermatozoa mL-1 while seminal plasma osmolality was 260±7.3-mOsm-kg-1. The percentage of viable sperm cells was determined with 97.0±2.0% and 87.0±8.0% using an eosin-nigrosin staining and fluorescent live-dead kit (propidium iodide and SYBR-14), respectively. A negative correlation (-0.65) between semen pH and sperm motility was observed suggesting this feature might influence the tambaqui sperm kinetics. Morphometrically the spermatozoa were on average 35.48±1.55μm long with a roundish head (mean length: 2.73±0.21μm; mean width: 2.58±0.18μm; n=250 spermatozoa), without an acrosome, and presented a long midpiece (2.90±0.52μm) and flagellum (29.84±1.63μm). About 15.8% of the spermatozoa carried morphological abnormalities, with bent tail being the most frequent defect (7.81±3.12%). The characterization of tambaqui semen favors the development of more precise and efficient procedures for its analysis and utilization in controlled breeding. © 2010 Blackwell Verlag, Berlin.

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