This study aimed to investigate the ability of disulphide-less crotamine (dLCr) to complex DNA and to evaluate whether the DNA–dLCr complex is capable of improving transfection in bovine embryos. Three experiments were performed to: (i) evaluate the formation and stability of the DNA–dLCr complex; (ii) assess the dLCr embryotoxicity by exposure of bovine embryos to dLCr; and (iii) assess the efficiency of bovine embryo transfection after microinjection of the DNA–dLCr complex or green fluorescent protein (GFP) plasmid alone (control). DNA complexation by dLCr after 30 min of incubation at 1:100 and 1:50 proportions presented higher efficiency (P < 0.05) than the two controls: native crotamine (NCr) 1:10 and lipofectamine. There was no difference between DNA–dLCr 1:25 and the controls. The DNA–dLCr complexation was evaluated at different proportions and times. In all, at least half of maximum complexation was achieved within the initial 30 min. No embryotoxicity of dLCr was verified after exposure of in vitro fertilized embryos to different concentrations of the peptide. The effectiveness of dLCr to improve exogenous gene expression was evaluated by microinjection of the DNA–dLCr complex into in vitro fertilized zygotes, followed by verification of both embryo development and GFP expression. From embryos microinjected with DNA only, 4.6% and 2.8% expressed the GFP transgene at day 5 and day 7, respectively. The DNA–dLCr complex did not increase the number of GFP-positive embryos. In conclusion, dLCr forms a complex with DNA and its application in in vitro culture is possible. However, the dLCr peptide sequence should be redesigned to improve GFP expression.

The present study investigated the effects of crotamine, a cell-penetrating peptide from rattlesnake venom, at different exposure times and concentrations, on both developmental competence and gene expression (ATP1A1, AQP3, GLUT1 and GLUT3) of in vitro fertilized (IVF) bovine embryos. In Experiment 1, presumptive zygotes were exposed to 0.1 μM crotamine for 6, 12 or 24 h and control groups (vehicle and IVF) were included. In Experiment 2, presumptive zygotes were exposed to 0 (vehicle), 0.1, 1 and 10 μM crotamine for 24 h. Additionally, to visualize crotamine uptake, embryos were exposed to rhodamine B-labelled crotamine and subjected to confocal microscopy. In Experiment 1, no difference (P > 0.05) was observed among different exposure times and control groups for cleavage and blastocyst rates and total cells number per blastocyst. Within each exposure time, mRNA levels were similar (P > 0.05) in embryos cultured with or without crotamine. In Experiment 2, concentrations as high as 10 μM crotamine did not affect (P > 0.05) the blastocyst rate. Crotamine at 0.1 and 10 μM did not alter mRNA levels when compared with the control (P > 0.05). Remarkably, only 1 μM crotamine decreased both ATP1A1 and AQP3 expression levels relative to the control group (P < 0.05). Also, it was possible to visualize the intracellular localization of crotamine. These results indicate that crotamine can translocate intact IVF bovine embryos and its application in the culture medium is possible at concentrations from 0.1–10 μM for 6–24 h.

Heavy metals are omnipresent in the environment, and industrial use has greatly increased their presence in soil, water and air. Their inevitable transfer to the human food chain remains an important environmental issue as many heavy metals cause a range of toxic effects, including developmental toxicity. Administration of chromium VI (1 and 2 mg/kg as potassium dichromate) through intraperitoneal (i.p.) injection during organogenesis (days 6 to 15 of gestation) in rats revealed embryo- and fetotoxic effects. Reduced fetal weight, retarded fetal development, number of fetuses per mother and high incidences of dead fetuses and resorptions in treated mothers were also observed. Gross morphological abnormalities, such as displayed form of edema, facial defect, lack of tail, hypotrophy, severs subdermal haemorrhage patches and hypotrophy of placenta were observed in fetuses after chromium VI-treated mothers. A skeletal development of fetuses presented an incomplete ossification in nasal, cranium, abdominal or caudal bones in rats treated with 1 mg/kg of chromium, whereas rats treated with 2 mg/kg showed ossification and absence of the sacral vertebrae compared with the control. At a higher dose of chromium, histological changes were found in fetuses with atrophy of theirs vital organs. Placental histological observations revealed a pronounced morphological alteration, with atrophy of decidual cells, a degenerated of chorionic villi and hypertrophy of blood lacuna. The present study suggests a risk to the developing embryo when the mother is exposed to a high concentration of chromium VI during organogenesis.