Trans-10,cis-12 conjugated linoleic acid reduces neutral lipid content and may affect cryotolerance of in vitro-produced crossbred bovine embryos

Background：Due to high neutral lipids accumulation in the cytoplasm,in vitro-produced embryos from Bos primigenius indicus and their crosses are more sensitive to chilling and cryopreservation than those from Bos primigenius taurus.The objective of the present study was to evaluate the effects of trans-10,cis-12 conjugated linoleic acid（CLA） on the development and cryotolerance of crossbred Bos primigenius taurus x Bos primigenius indicus embryos produced in vitro,and cultured in the presence of fetal calf serum.Bovine zygotes（n = 1,692）were randomly assigned to one of the following treatment groups：1） Control,zygotes cultured in Charles Rosenkrans 2 amino acid（CR2aa） medium（n = 815） or 2） CLA,zygotes cultured in CR2 aa medium supplemented with 100 μmol/L of trans-10,cis-12 CLA（n =877）.Embryo development（cleavage and blastocyst rates evaluated at days 3 and 8 of culture,respectively）,lipid content at morula stage（day 5） and blastocyst cryotolerance（re-expansion and hatching rates,evaluated 24 and 72 h post-thawing,respectively） were compared between groups.Additionally,selected mRNA transcripts were measured by Real-Time PCR in blastocyst stage.Results：The CLA treatment had no effect on cleavage and blastocyst rates,or on mRNA levels for genes related to cellular stress and apoptosis.On the other hand,abundance of mRNA for the 1-acylglycerol-3-phosphate0-acyltransferase-encoding gene（AGPAT）,which is involved in triglycerides synthesis,and consequently neutral lipid content,were reduced by CLA treatment.A significant increase was observed in the re-expansion rate of embryos cultured with trans-10,cis-12 CLA when compared to control（56.3 vs.34.4%,respectively,P = 0.002）.However,this difference was not observed in the hatching rate（16.5 vs.14.0%,respectively,P=0.62）.Conclusions：The supplementation with trans-10,ds-12 CLA isomer in culture medium reduced the lipid content of in vitro produced bovine embryos by reducing the gene expression of 1-acylglycerol 3-phosphate 0-acyltransferase（AGPAT） enzyme.However,a possible improvement in embryo cryotolerance in response to CLA,as suggested by increased blastocyst re-expansion rate,was not confirmed by hatching rates.

Challenges for the Characterization of Genetically Modified Animals by the qPCR Technique in the Era of Genomic Editing

Characterization of genetically modified organisms through determina­tion of zygosity and transgene integration concerning both copy number and genome site is important for breeding a transgenic line and the use of these organisms in the purpose for which it was obtained.Southern blot,fluorescence in situ hybridization or mating are demanding and time-consuming techniques traditionally used in the characterization of transgenic organisms and,with the exception of mating,give ambiguous results.With the emergence of the real-time quantitative PCR technology,different applications have been described for the analysis of transgenic organisms by determination of several parameters to transgenic analysis.However,the accuracy in quantitation by this method can be influenced in all steps of analysis.This review focuses on the aspects that influence pre-analytical steps(DNA extraction and DNA quantification methods),quantification strategies and data analysis in quantification of copy num­ber and zygosity in transgenic animals.