ABSTRACT
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.
Subject(s)
Crotalid Venoms/pharmacology , DNA/chemistry , Disulfides/chemistry , Embryo, Mammalian/physiology , Fertilization in Vitro/veterinary , Peptide Fragments/chemistry , Transfection/methods , Animals , Cattle , Cells, Cultured , Crotalid Venoms/chemistry , DNA/metabolism , Embryo, Mammalian/cytology , Embryo, Mammalian/drug effects , Female , Gene Transfer Techniques , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Male , Oocytes/cytology , Oocytes/drug effects , Oocytes/physiology , Peptide Fragments/metabolismABSTRACT
SummaryThis study aimed to compare the efficiency of different incubation systems for in vitro embryo production in bovine. Oocytes/embryos were cultured in three incubators: conventional - CONV, mini bench - MINI and portable - PORT. After in vitro maturation (IVM), oocytes were verified for maturation rate. The remaining structures were submitted to in vitro fertilization and culture to verify cleavage (day 2) and blastocyst (day 7) rates. Reactive oxygen species (ROS) were evaluated in post-IVM oocytes and embryos (days 2 and 7) using arbitrary fluorescence units (AFUs). No significant difference (P>0.05) was observed for maturation rate. The CONV system (74.0%) produced the highest cleavage rate (P0.05) to MINI (65.0%). The same pattern and differences were observed for blastocyst rate: CONV (33.3%), MINI (32.3%) and PORT (21.9%). ROS levels were not different (P>0.05) in post-IVM oocytes: CONV (35.6±4.5), MINI (29.4±4.0) and PORT (35.6±4.5). For day-2 embryos, ROS levels were higher (P0.05) was observed in blastocysts. In conclusion, although it produced high ROS levels at day 2 of culture, the MINI system was as efficient as the CONV system for blastocyst production. This option may be an interesting and economical for the in vitro embryo industry.
Subject(s)
Embryonic Development , Fertilization in Vitro/veterinary , In Vitro Oocyte Maturation Techniques , Incubators/veterinary , Oocytes/physiology , Animals , Cattle , Female , Fertilization in Vitro/instrumentation , Fertilization in Vitro/methods , Oocytes/cytologyABSTRACT
The aim of this study was to evaluate the use of antifreeze protein type III (AFP III) into vitrification medium on meiotic spindle morphology of in vitro matured bovine oocytes as well as the fertilization and blastocyst rates. Mature cumulus-oocyte complexes (COC) were distributed in four groups: control (untreated), vitrified without supplementation (AFP0) or supplemented with 500 (AFP500) or 1000 ng/mL (AFP1000) into vitrification solutions. Samples from each group were used to analyze the organization of meiotic spindle by confocal microscopy and the remaining COC were submitted to in vitro fertilization and culture for eight days. Control group exhibited only 15% of abnormal spindle. However, the spindle morphology was affected in all vitrified groups regardless to AFP concentration: 75.8%, 76.1% and 69.2% (P > 0.05) for AFP0, AFP500 and AFP1000, respectively. Similar cleavage rate was obtained among the vitrified groups (AFP0 = 17.9%, AFP500 = 16.9% and AFP1000 = 17.8%), but lower (P < 0.05) compared with control group (68.7%). At Day 5 of culture, embryo production rate of AFP500 (30.8%) and AFP1000 (25.0%) were similar to control group (49.4%). However, at Day 8 of culture, AFP0, AFP500 and AFP1000 groups exhibited lower (P < 0.05) blastocyst rates (10.0%, 3.8% and 9.4%, respectively) when compared to control (41.1%). In conclusion, AFP III did not preserve meiotic spindle organization against the cryoinjuries. However, the use of AFP III improved embryo development at Day 5 of culture, although this effect was not maintained up to the blastocyst formation.
