No abstract

Our results demonstrate that the addition of Vitamin E may enhance the developmental competence of buffalo embryos in vitro by protecting them from oxidative stress.

Vitamin A is a well-known antioxidant and is essential for embryonic development, growth and differentiation. Oxidative stress is involved in the etiology of defective embryo development. The present study evaluated whether the presence of all-trans retinol (0, 1, 2, 5 and 10 mM) in maturation medium or embryo culture medium would enhance the developmental competence of preimplantation buffalo embryos in vitro. In experiment I, cumulus oocytes complex were matured with varying concentrations of all-trans retinol. Treatment with 5 mM all-trans retinol improved the blastocyst formation ( P , 0.001) when compared with control and significant increase ( P , 0.01) in total cell number was observed in 5 mM group when compared with control. Supplementation of all-trans retinol in embryo culture medium for the entire culture period under 5% O 2 and 20% O 2 was tested in experiments II and III, respectively. Supplementation of 10 mM all-trans retinol under 5% O 2 , significantly reduced blastocyst formation and cell numbers. Presence of 5 mM all-trans retinol under 20% O 2 enhanced the frequency of blastocyst formation and total cell number ( P , 0.001) when compared with control. DNA damage of individual embryos cultured under 20% oxygen concentration was measured by the comet assay. Supplementation of 5 mM all-trans retinol significantly reduced the comet tail ( P , 0.001) when compared with control. Supplementation of all-trans retinol in embryo culture medium for first 72 h of the 8-day culture period under 5% O 2 was tested in experiment IV. Addition of 5 mM all-trans retinol resulted in significant increase in blastocyst rate and total cell number ( P , 0.001) when compared with control. Our results demonstrate that addition of all-trans retinol to maturation or embryo culture medium may enhance the developmental competence of buffalo embryos in vitro by enhancing blastocyst formation rate and total cell number.

Tyrosine hydroxylase and phenoloxidase differ in that tyrosine hydroxylase (E.C.1.14.16.2) can hydroxylate tyrosine into -o-diphenol, but cannot oxidize the -o-diphenol, whereas phenoloxidase (E.C.1.14.18.1) is capable of oxidizing -o-diphenol to quinone. This difference can be exploited by staining tyrosine hydroxylase activity with a substrate-PMS-NBT method and staining the phenoloxidase with a dopamine-MBTH method. Based on the staining properties of the bands separated after electrophoresis, tyrosine hydroxylase has been differentiated from phenoloxidase in the silkworm Bombyx mori and the occurrence of tyrosine hydroxylase has been reported for the first time in this worm.