GC-(reverse) for glyceraldehyde--phosphate dehydrotials and found to be biosynthesized di erentially during chain reaction (PCR) of chromo-helicase DNA binding developmental stages in male and female avian embryos protein described by Fridolfsson and Ellegren ().(Scheib). The sequence of the primer sets were-GTT ACT GAT To our knowledge there is no report on the infiuence of TCG TCT ACG AGA-(forward) and-ATT GAA developmental stages and sexes of avian embryo to metalinduced toxicity. The present study was designed to consisted of total cycles with-min initial denaturizing investigate the in-ovo exposure e ects of Cd on the embry- at followed by-min denaturing at,-min onic survivability and hatchling weight of Japanese quail. annealing at and-min extension at. The The sex specificity of developing quail embryo to Cd amplified PCR products from male embryonic DNA protoxicity and ascorbic acid amelioration have also been duced a single band, whereas a double band was found in evaluated by monitoring some well-known stress indica- case of female embryos after separation in agarose gel tors like MT mRNA expression, catalase activity, and electrophoresis. malondialdehyde (MDA) production. The total RNA were extracted from the liver of dayembryo by RNAiso (Takara, Japan) following the manufacturer’s protocol. The concentration and purity of Fertile eggs from a breeding stock of-to-week-old RNA were determined by measuring the light absorption Japanese quail were used in the experiments. The intact at and nm using a spectrophotometer (Gene shelled eggs weighing approximately to g were col- Quant; Pharmacia Biotech, Uppsala, Sweden). RNA lected and stored at up to days before setting into samples exhibiting an A to A ratio of. or more were incubator. The CdCl and L ()-ascorbic acid were used for the next step of the experiment (Sambrook and purchased from Wako Pure Chemical (Osaka, Japan) and Russell,). used as the test agents. All other chemicals including the MT mRNA expressions in the day-embryonic liver thiobarbituric acid and,,,-tetraethoxypropane (TEP) were determined using a semi-quantitative RT-PCR prowere of the reagent grade and purchased from Wako Pure tocol of Rahman(). In brief, g of total RNA Chemical. was reverse-transcribed to cDNA following to the protocol of SuperScript First-Strand Synthesis System for Cd and ascorbic acid were injected into the fertile eggs just before setting into the incubator through a hole primer. The genomic DNA-contaminated RNA samples making on the shell with a-gauge needle and putting were avoided by RT-negative test. Then, the cDNA was them approximately at the center of the yolk. A volume of amplified by PCR with the primer sets of-TCA GGA sterile saline/egg was used as the vehicle of Cd or CTG CAC TTG TGC TGC-(forward) and-TGG ascorbic acid. In case of co-exposure of Cd and ascorbic CAG CAG CTG CAC TTG CT-(reverse) for MT acid, a common saline solution carried both the(product size, base pairs; GenBank Accession No. chemicals together. After injection, the hole on the egg- AY) and-CCA GGG CTG CTG TCC TTT CT-shell was covered with a piece of mending tape.(forward) and-CAG AGA CAT TGG GGG TTG genase (GAPDH; product size, base pairs; GenBank The eggs were incubated in a force-draft and auto- Accession No. Z). T turning electronic incubator (Showa Furanki, Japan). A constant temperature of with relative humidity was maintained in the incubator throughout the incubation period. The infertility and early embryonic death in the Cd-injected egg were checked at day-and day-of incubation by candling with an …