Buffalo Embryos Produced by Handmade Cloning from Oocytes Selected Using Brilliant Cresyl Blue Staining Have Better Developmental Competence and Quality and Are Closer to Embryos Produced byIn VitroFertilization in Terms of Their Epigenetic Status and Gene Expression Pattern

Abstract

We compared handmade cloned (HMC) buffalo blastocysts produced from oocytes stained with Brilliant Cresyl Blue (BCB) and classified into those with blue (BCB + ) or colorless cytoplasm (BCB -). The blastocyst rate was higher ( p < 0.001) for BCB + than for BCB -oocytes (43.41 -2.54 vs. 22.74 -1.76%). BCB + blastocysts had inner cell mass (ICM) cell number, ICM-to-trophectoderm ratio, global level of H3K18ac, apoptotic index, and expression level of BCL-XL, but not that of CASPASE-3, similar to that of blastocysts produced through in vitro fertilization (IVF), which was higher ( p < 0.05) than that of BCB -blastocysts. The global level of H3K9me2, which was similar in BCB + and BCB -blastocysts, was higher ( p < 0.01) than that in IVF blastocysts. The expression level of OCT4 and SOX2 was higher ( p < 0.05) and that of GATA2 was lower ( p < 0.05) in BCB + than that in BCB -blastocysts, whereas that of DNMT1, DNMT3a, NANOG, and CDX2 was not significantly different between the two groups. The expression level of DNMT1, OCT4, NANOG, and SOX2 was lower ( p < 0.05) and that of CDX2 was higher ( p < 0.05) in BCB + than in IVF blastocysts. In conclusion, because BCB + blastocysts have better developmental competence and are closer to IVF blastocysts in terms of quality, epigenetic status, and gene expression than BCB -blastocysts, BCB staining can be used effectively for selection of developmentally competent oocytes for HMC.

RNA sequencing and transcriptome analysis of buffalo (Bubalus bubalis) blastocysts produced by somatic cell nuclear transfer and in vitro fertilization

“…Compact COCs which possessed evenly granular homogenous ooplasm and unexpanded cumulus mass with two or more layers of cumulus cells were classified as usable whereas, those which were wholly or partially denuded, or had an expanded cumulus mass or irregular ooplasm were classified as nonusable. Staining with brilliant cresyl blue (BCB), as described previously (Mohapatra et al, ), was used for selection of COCs of high developmental competence. The COCs were then washed three times with dulbecco's phosphate‐buffered saline (DPBS) and those with distinct blue color (BCB+) were washed several times with the IVM medium, which consisted of TCM‐199 + 10% FBS + 5 µg/ml porcine follicle‐stimulating hormone + 1 µg/ml estradiol‐17β + 0.81 mM sodium pyruvate + 10% buffalo follicular fluid + 50 µg/mL gentamicin sulfate.…”

Buffalo SCNT embryos exhibit abnormal gene expression of ERK/MAPK pathway and DNA methylation

“…An abnormally increased expression of Dnmt1 protein has also been observed in the 8‐cell stage cloned murine embryos (Chung, Ratnam, Chaillet, & Latham, ). In a recent study, we found the expression level of DNMT1 to be higher in cloned buffalo blastocysts compared to IVF counterparts (Mohapatra, Sandhu, Neerukattu, et al, ; Mohapatra, Sandhu, Singh, et al, ). DNA hypermethylation, as a consequence of high activity of DNMTs in cloned embryos, is an important epigenetic aberration not only because it directly results in gene repression but also because it influences histone modifications although both are carried out by different chemical reactions (Cedar & Bergman, ).…”

“…Somatic cells obtained from ear skin biopsy taken from an adult buffalo bull (Mu‐5926), which had already been established and characterized in the laboratory (Selokar et al, ), were used as donor cells for HMC for producing cloned blastocysts. Cumulus‐oocyte complexes (COCs) collected from slaughterhouse buffalo ovaries were subjected to staining with Brilliant Cresyl Blue (BCB) for selection of developmentally competent oocytes as described earlier (Mohapatra, Sandhu, Neerukattu, et al, ; Mohapatra, Sandhu, Singh, et al, ). HMC, which included in vitro maturation (IVM), cumulus/zona removal, manual enucleation, fusion, activation and in vitro culture (IVC), was performed as described earlier (Selokar et al, ).…”

“…The relative expression level of various genes was determined in cloned and IVF blastocysts by qPCR using GAPDH (Mohapatra, Sandhu, Neerukattu, et al, ; Mohapatra, Sandhu, Singh, et al, ) and β‐actin (Saini et al, ; Selokar et al, ) as internal control genes. Primer sequences were designed using Primer express 3.0 software (Applied Biosystem) ensuring through BLAST that they did not show significant homology to other sequences in the genome.…”

Effects of oocyte source, cell origin, and embryo reconstruction procedures on in vitro and in vivo embryo survival after goat cloning

“…The oocyte quality and competence play a crucial role in the success of a cloning program, since the ooplasm is responsible for reprogramming the nucleus donor, which has an important effect on subsequent development (Fissore et al, 1999;Kelly et al, 2007;Mohapatra et al, 2015). Normally, better quality oocytes, usually grades I (GI) and II (GII), are selected for in vitro maturation (Chen et al, 2007;Tang et al, 2011).…”