Comparative study of NiO/CuO/Ag doped graphene based materials for reduction of nitroaromatic compounds and degradation of dye with statistical study.

In the present work, the Nickel oxide (rGO-NiO), Silver (rGO-Ag), Copper oxide (rGO-CuO) doped Graphene Oxide are reported for catalytic reactions. A comparative study for catalytic activities of these materials are performed with nitroaromatic compound 4-nitroaniline and the results are statistically studied by using univariate analysis of variance and Post Hoc Test through Statistical Package for Social Sciences and it is observed that CuO doped Graphene material is showing better catalytic activity in minimum time. So, further research has been focused on the catalytic acitivity of rGO-CuO only and it is found that it is efficient in reducing other nitro compounds also such as Picric acid and Nitrobenzene. Dye degradation of Methylene blue is also performed using CuO decorated Graphene material and significant changes were observed using UV spectroscopy. The characterization of rGO-CuO is done with Fourier-transform Infrared Spectroscopy, Powder X-ray Diffraction, Thermogravimetric Analysis, Scanning Electron Microscope and Transmission Electron Microscopy.

Reduced graphene Oxide/NiO based nano-composites for the efficient removal of alizarin dye, indigo dye and reduction of nitro aromatic compounds.

Removal of alizarin red S (ARS) and Indigo dye from aqueous media and reduction of nitro aromatic compounds are successfully done under mild condition by using reduced Graphene Oxide-Nickel Oxide (rGO-NiO) nanocomposite as catalyst. RGO-NiO is well characterized by different analytical techniques. Morphology, structural, and composition studies done by HRTEM, FESEM, EDX, TGA, FTIR, XPS, Raman spectroscopy, and XRD. RGO-NiO nanocomposite has high stability for the removal of ARS, Indigo dye, reduction reaction nitro aromatic compounds.

Sequence characterization and comparative expression profile of buffalo WNT10B gene in adult and fetal tissues.

In this study, Wingless-type MMTV (mouse mammary tumor virus) integration site family member (WNT10B) gene was sequence characterized in the Indian water buffalo. Sequence analysis revealed an open reading frame of 1176 nucleotides in buffalo, encoding 391 amino acids long protein. Nineteen nucleotide variations were observed between cattle and buffalo resulting in six amino acid changes. Phylogenetic analysis showed the clustering of ruminant species together. Real-time expression analysis of WNT10B in tissues collected from different organs of fetal and adult buffalo, revealed, the gene being abundantly expressed in the rumen and liver of the fetus. The fetal ovary, heart, kidney, lung, testis and mammary gland showed moderate expression, while in adult tissues, expression was high in the ovary, testis, brain, kidney, small intestine and liver, whereas lower expression was observed in the adult rumen. Significant differences in WNT10B expression levels were found for the brain, small intestine, testes, kidney, heart, rumen, and ovary when adult and fetal tissues were compared. A moderate level of genetic variation was found between cattle and buffalo WNT10B and expression patterns in a variety of tissues in adult buffalo implies that in addition to possible roles in adipogenesis and hematopoiesis, the WNT10B gene might be playing a significant role in other regulatory pathways as well.

Propagation of porcine spermatogonial stem cells in serum-free culture conditions using knockout serum replacement.

In vitro culture and expansion of spermatogonial stem cells (SSCs) is an essential prerequisite to enhancing livestock productivity through SSC transplantation. Most of the culture media have been observed to be supplemented with serum. However, the use of serum in culture media may exert detrimental effects on SSC maintenance in vitro. An attempt was made to culture SSCs by replacing serum with 5% 'Knockout Serum Replacement (KSR)' in Doom pig (Sus domesticus), one of the valued indigenous germplasm of North-East India. Testes from 7 to 15 days old piglets were used for isolation, enrichment and in vitro culture of putative SSCs using serum-based and serum-free culture media. The cells were characterized for SSC-specific pluripotent markers expression by immunofluorescence staining and quantitative real-time PCR. The diameter and number of SSC colonies were recorded on days 9, 20 and 30 of culture. Similar morphologies of the SSC colonies were observed in both serum-based and serum-free culture conditions. Colony diameter and colony number were non-significantly higher in serum-free than serum-based media. The cells from both the culture conditions showed high alkaline phosphatase activity. The expression of SSC-specific pluripotent markers was observed in immunofluorescence and quantitative real-time PCR study. The present study revealed that SSCs from porcine species could be maintained in vitro for up to 30 days in serum-free culture using 5% KSR, which is believed to be a promising protein source for improving livestock production, and health care along with their conservation.

Recent Trends in Photocatalytic Enantioselective Reactions.

Enantioselective synthesis through photocatalysis is one of the highly preferred approaches towards preparation of optically active compounds. This review elaborates and critically analyzes the different strategies of photocatalytic enantioselective reactions through H-bonding, transition metal catalysis, phase-transfer catalysis (PTC), chiral Lewis acid catalysis, N-heterocyclic carbene catalysis, and amine catalysis, and also explores ion pairs. In addition, it explains the different catalysis modes with multifunctional approaches for enantioselective photocatalytic reactions.

Development of homologous hemizona assay as a potential predictor of fertility in buffalo bulls: A preliminary approach.

Although, myriads of tests are routinely used, no single test can accurately predict fertilization potential of semen. The hemizona assay (HZA) has advantages in two ways: (a) it determines multitude traits of sperm and (b) it is a controlled sperm function test. In the present study, we developed homologous HZA in buffaloes to predict bull fertility. In this experiment, bulls with fertility rate 53.3% and 48.5% were used as control, whereas bulls with fertility rate 32.6% and 32.2% were used as test semen samples. For HZA, matching buffalo hemizonae were co-incubated with processed buffalo sperm for 4 h. The number of sperms bound to the outer surface of hemizona was determined. No significant difference was observed in sperm binding for co-incubation of same bull sperm with matching hemizona (p < .05). Significant difference in sperm binding to matching hemizona was seen, while two halves were incubated with control and test semen, respectively (p < .05). Hemizona assay index (HZAI) of test bull semen has been determined from percentage of test-sperm bound to the matching hemizona in comparison to control-sperm. For finding relation, HZAI was correlated with respective fertility rates of semen samples, and it was found that a significant positive correlation was present with r = 0.83, p < .1. A regression equation of Y = 1.39X - 55.8 (where Y = pregnancy rate of test semen sample and X = HZAI of test semen sample) was presented to predict fertility rates of unknown semen samples. Thus, HZA can be used as a potential predictor of buffalo bull fertility.

Synthesis of N-Benzylideneaniline by Schiff base reaction using Kinnow peel powder as Green catalyst and comparative study of derivatives through ANOVA techniques.

The cheap and easy availability of the Kinnow peel waste has reported various applications due to presence of multifunctional groups. Therefore, in present study we explored its application to synthesize N-Benzylideneaniline and its derivatives based on Schiff base reaction. Kinnow peel powder is characterized by FTIR, TEM, SEM, XRD, EDX, and TGA for functional groups, morphology, surface, elements and thermal stability. Benzaldehyde, aniline, and their derivatives such as 4-methyl benzaldehyde, 4-hydroxy benzaldehyde, 4-methoxy benzaldehyde, and 4-methoxy aniline have been used to compare the efficacy of the Schiff base reaction using analysis of variance (ANOVA) and it has been observed that combination of Aniline and benzaldehyde for Schiff base reaction provided 85% yield of relative product.

Reproductive stage- and season-dependent culture characteristics of enriched caprine male germline stem cells.

The present study aims to evaluate season- and reproductive-stage dependent variation in culture characteristics and expression of pluripotency and adhesion markers in caprine-male germline stem cells (cmGSCs). For this, testes from pre-pubertal (4-6 months) and adult (~ 2 years) bucks during non-breeding (July-August; n = 4 each) and breeding (October-November; n = 4 each) seasons were used to isolated testicular cells by two-step enzymatic digestion. After cmGSCs enrichment by multiple methods (differential platting, Percoll density gradient centrifugation, and MACS), cell viability of CD90+ cells was assessed before co-cultured onto the Sertoli cell feeder layer up to 3rd-passage (P-3). The culture characteristics of cmGSCs were compared during primary culture (P-0) and P-3 with different assays [BrdU-assay (proliferation), MTT-assay (senescence), and Cluster-forming activity-assay] and transcript expression analyses by qRT-PCR. Moreover, the co-localization of UCHL-1, CD90, and DBA was examined by a double-immunofluorescence method. In adult bucks, significantly (p < 0.05) higher cell numbers with the ability to proliferate faster and form a greater number of cell clusters, besides up-regulation of pluripotency and adhesion markers expression were observed during the breeding season than the non-breeding season. In contrast, such season-dependent variation was lacking in pre-pubertal bucks. The expression of transcripts during non-breeding seasons was significantly (p < 0.05) higher in pre-pubertal cmGSCs than in adult cells (UCHL-1 = 2.38-folds; CD-90 = 6.66-folds; PLZF = 20.87-folds; ID-4 = 4.75-folds; E-cadherin = 3.89-folds and ß1-integrin = 5.70-folds). Overall, the reproductive stage and season affect the population, culture characteristics, and expression of pluripotency and adhesion specific markers in buck testis. These results provide an insight to develop an efficient system for successful cell culture processes targeting cmGSCs. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00515-x.

Relative expression of the developmentally important candidate genes in immature oocytes and in vitro-produced embryos of buffalo (Bubalus bubalis).

The study was undertaken to examine the relative abundance (RA) of the major developmental important candidate genes in different grades of immature oocytes (A-grade, B-grade, C-grade and D-grade) and various stages of in vitro-produced embryos (2-cell, 4-cell, 8-16-cell, morula, and blastocyst) of buffalo using RT-qPCR. Results showed that the RA of GLUT1, CX43, HSP70.1 and GDF9 was significantly higher (P < 0.05) in the A-grade of oocytes than the C-grade and D-grade but did not differ significantly from the B-grade of oocytes. Similarly, RA of BMP15 and Survivin were significantly higher (P < 0.05) in A-grade than the other grades of oocytes, however, poly(A) polymerase expression was not significantly different (P > 0.05) among the immature oocytes. The expression of GLUT1 was significantly higher (P < 0.05) in the blastocysts, but the expression of CX43 (P < 0.05; P > 0.05), HSP70.1 (P < 0.05; P > 0.05) and GDF9 (P > 0.05) was higher at the 2-cell stage than the other stages of embryos. Interestingly, the expression levels of poly(A) polymerase (P < 0.05), BMP15 (P < 0.05; P > 0.05) and Survivin (P > 0.05) were higher at the 8-16-cell stage than the other stages of embryos. It is concluded that A-grade of immature oocytes has shown more mRNA abundance for the major developmental important genes; therefore A-grade oocytes may be considered as the most developmentally competent and suitable for handmade cloning research in buffalo.

Exposure to Pulsed Electromagnetic Fields Improves the Developmental Competence and Quality of Somatic Cell Nuclear Transfer Buffalo (Bubalus bubalis) Embryos Produced Using Fibroblast Cells and Alters Their Epigenetic Status and Gene Expression.

We examined the effects of treatment with pulsed electromagnetic fields (PEMFs) on cumulus cells and buffalo somatic cell nuclear transfer (SCNT) embryos. PEMF treatment (30 µT for 3 hours) of cumulus cells increased (p < 0.05) the relative cell viability and cell proliferation and the expression level of OCT4, NANOG, SOX2, P53, CCNB1, and GPX, but decreased (p < 0.05) that of DNMT1, DNMT3a, GSK3b, and BAX, whereas the expression level of DNMT3b, GLUT1, BCL2, CASPASE3, SOD1, and CATALASE was not affected. PEMF treatment of SCNT embryos at the beginning of in vitro culture increased (p < 0.05) the blastocyst rate (51.4% ± 1.36% vs. 42.8% ± 1.29%) and decreased (p < 0.01) the apoptotic index to the level in in vitro fertilization blastocysts, but did not significantly alter the total cell number and the inner cell mass:trophectoderm cell number ratio of blastocysts compared to the controls. PEMF treatment increased the expression level of NANOG, SOX2, CDX2, GLUT1, P53, and BCL2 and decreased that of BAX, CASPASE3, GSK3b, and HSP70, but not OCT4, DNMT1, DNMT3a, DNMT3b, HDAC1, and CCNB1 in blastocysts. It increased (p < 0.001) the global level of H3K27me3 but not H3K18ac. These results suggest that PEMF treatment of SCNT embryos improves their developmental competence, reduces the level of apoptosis, and alters the expression level of several important genes related to pluripotency, apoptosis, metabolism, and stress.

Production of Transgenic Handmade Cloned Goat (Capra hircus) Embryos by Targeted Integration into Rosa 26 Locus Using Transcription Activator-like Effector Nucleases.

Transgenic goats are ideal bioreactors for the production of therapeutic proteins in their mammary glands. However, random integration of the transgene within-host genome often culminates in unstable expression and unpredictable phenotypes. Targeting desired genes to a safe locus in the goat genome using advanced targeted genome-editing tools, such as transcription activator-like effector nucleases (TALENs) might assist in overcoming these hurdles. We identified Rosa 26 locus, a safe harbor for transgene integration, on chromosome 22 in the goat genome for the first time. We further demonstrate that TALEN-mediated targeting of GFP gene cassette at Rosa 26 locus exhibited stable and ubiquitous expression of GFP gene in goat fetal fibroblasts (GFFs) and after that, transgenic cloned embryos generated by handmade cloning (HMC). The transfection of GFFs by the TALEN pair resulted in 13.30% indel frequency at the target site. Upon cotransfection with TALEN and donor vectors, four correctly targeted cell colonies were obtained and all of them showed monoallelic gene insertions. The blastocyst rate for transgenic cloned embryos (3.92% ± 1.12%) was significantly (p < 0.05) lower than cloned embryos (7.84% ± 0.68%) used as control. Concomitantly, 2 out of 15 embryos of morulae and blastocyst stage (13.30%) exhibited site-specific integration. In conclusion, the present study demonstrates TALEN-mediated transgene integration at Rosa 26 locus in caprine fetal fibroblasts and the generation of transgenic cloned embryos using HMC.

Low oxygen tension potentiates proliferation and stemness but not multilineage differentiation of caprine male germline stem cells.

The milieu of male germline stem cells (mGSCs) is characterized as a low-oxygen (O2) environment, whereas, their in-vitro expansion is typically performed under normoxia (20-21% O2). The comparative information about the effects of low and normal O2 levels on the growth and differentiation of caprine mGSCs (cmGSCs) is lacking. Thus, we aimed to investigate the functional and multilineage differentiation characteristics of enriched cmGSCs, when grown under hypoxia and normoxia. After enrichment of cmGSCs through multiple methods (differential platting and Percoll-density gradient centrifugation), the growth characteristics of cells [population-doubling time (PDT), viability, proliferation, and senescence], and expression of key-markers of adhesion (ß-integrin and E-Cadherin) and stemness (OCT-4, THY-1 and UCHL-1) were evaluated under hypoxia (5% O2) and normoxia (21% O2). Furthermore, the extent of multilineage differentiation (neurogenic, adipogenic, and chondrogenic differentiation) under different culture conditions was assessed. The survival, viability, and proliferation were significantly (p < 0.05) improved, thus, yielding a significantly (p < 0.05) higher number of viable cells with larger colonies under hypoxia. Furthermore, the expression of stemness and adhesion markers were distinctly upregulated under lowered O2 conditions. Conversely, the differentiated regions and expression of differentiation-specific genes [C/EBPα (adipogenic), nestin and ß-tubulin (neurogenic), and COL2A1 (chondrogenic)] were significantly (p < 0.05) reduced under hypoxia. Overall, the results demonstrate that culturing cmGSCs under hypoxia augments the growth characteristics and stemness but not the multilineage differentiation of cmGSCs, as compared with normoxia. These data are important to develop robust methodologies for ex-vivo expansion and lineage-committed differentiation of cmGSCs for clinical applications.

Relative abundance of pluripotency-associated candidate genes in immature oocytes and in vitro-produced buffalo embryos (Bubalus bubalis).

The present study was undertaken to analyze the relative abundance (RA) of pluripotency-associated genes (NANOG, OCT4, SOX2, c-MYC, and FOXD3) in different grades of immature oocytes and various stages of in vitro-produced buffalo embryos using RT-qPCR. Results showed that the RA of NANOG, OCT4, and FOXD3 transcripts was significantly higher (P < 0.05) in A grade oocytes compared with the other grades of oocytes. The RA of the c-MYC transcript was significantly higher (P < 0.05) in A grade compared with the C and D grades of oocytes, but the values did not differ significantly from the B grade of oocytes. The RA of the SOX2 transcript was almost similar in all grades of the oocytes. The expression levels of NANOG (P > 0.05), OCT4 (P > 0.05), c-MYC (P > 0.05) and SOX2 (P < 0.05) were higher in the blastocysts compared with the other stages of the embryos. Markedly, FOXD3 expression was significantly higher (P < 0.05) in 8-16-cell embryos compared with the 2-cell and 4-cell embryos and blastocyst, but did not differ significantly from the morula stage of the embryos. In the study, the majority of pluripotency-associated genes showed higher expression in A grade immature oocytes. Therefore, it is concluded that the A grade oocytes appeared to be more developmental competent and are suitable candidates for nuclear cloning research in buffalo. In buffalo, NANOG, OCT4, SOX2, and c-MYC are highly expressed in blastocysts compared with the other stages of embryos.

Media switching at different time periods affects the reprogramming efficiency of buffalo fetal fibroblasts.

Many contrasting reports are available on generation of bovine induced pluripotent stem cells (iPSCs) employing different timelines and culture conditions which signifies reprogramming process varies between species and cell types. The present study determines an optimum time period required to re-initiate reprogramming events in buffalo fibroblasts after introduction of exogenous genes (OCT4, SOX2, KLF4 and c-MYC) by lentiviral vector. The reprogramming efficiency is cumulative result of many factors including culture conditions and addition of growth factors in culture media. In our study, we observed when stem cell culture conditions were provided Day 5 post-transduction, it results in maximum reprogramming efficiency in comparison when same conditions were provided too early or on later days. The putative iPSCs were expanded on feeder layer for 15 passages and found positive for alkaline phosphatase and pluripotency markers (OCT4, SOX2, KLF4, c-MYC, UTF, TELOMERASE, FOXD3, REX1, STAT3, NUCLEOSTAMIN and TRA1-81). Also, they produced embryoid bodies showing expression for ectodermal (NF68, MOBP), mesodermal (ASA, BMP4) and endodermal (GATA4, AFP) markers to confirm their pluripotent nature. Our results suggest that reprogramming is accompanied by time dependent events and providing stem cell culture conditions at definite time during reprogramming can help in generation of iPSCs with greater efficiency.

Optimization and Comparison of Three-Dimensional Culture Conditions in Different Media of Coculture and Encapsulation System for In Vitro Follicular Development in Bubalus bubalis.

The establishment of an in vitro culture system for complete oocyte maturation from the early stages of ovarian follicles is still a challenge. The aim of the present study was to assess the effect of different matrix with different culture media on the developmental growth of ovarian follicles in vitro. An ovarian histoarchitectural study was carried out to identify the primordial (0.027-0.039 mm), primary (0.041-0.079 mm), small preantral (0.085-0.131 mm), large preantral (0.132-0.294 mm), small antral (0.387-0.589 mm), and large antral (1.188-1.366 mm) follicles. Thus, large preantral follicles (0.2-0.3 mm) were mechanically isolated and cultured subsequently in different microconditions such as Dulbecco's modified Eagle's medium, Tissue Culture Medium-199 (TCM-199) and Opti-minimum essential medium, with same supplements where control (without matrix) was compared with matrix (coculture and encapsulation), which includes (1) buffalo fetal fibroblast cells, (2) cumulus cells, (3) ovarian mesenchymal cells, (4) collagen, (5) gelatin, and (6) Matrigel, cultured for 7 days in CO2 incubator at 38.5°C (5% CO2 in air). Cultured follicles were evaluated for growth rate (107.88% ± 10.24%), maturation rate (51.06% ± 6.53%), survivability rate (56.52% ± 3.42%), and antioxidant (catalase; CAT [1.58 ± 0.04 U/mg], superoxide dismutase; SOD [4.63 ± 0.05 U/mg], lactate dehydrogenase; LDH [1.48 ± 0.01 U/mg]) enzymatic activities, which showed significantly (p < 0.05) positive results in growth model with media TCM-199 than other studied groups. Furthermore, the development of large preantral follicles augmented significantly (p < 0.05) for growth rate (248.54% ± 9.51%), maturation rate (75.81% ± 7.07%), survivability rate (81.82% ± 3.02%), antioxidant (CAT [2.05 ± 0.03 U/mg], SOD [3.13 ± 0.12 U/mg], LDH [2.55 ± 0.51 U/mg]), and estradiol (175.83 ± 5.92 pg/mL) activities when they were encapsulated in Matrigel with nutritional requirements fulfilled by media TCM-199. These results provide better insight for the optimization of culture conditions for in vitro follicular development in the water buffalo, which will eventually assist in resolving the limitation of obtaining fewer competent oocytes for the embryo production in the species.

Effect of basic fibroblast growth factor (FGF2) on cumulus cell expansion, in vitro embryo production and gene expression in buffalo (Bubalus bubalis).

The present study was undertaken to evaluate the effect of different concentration of FGF2 viz. 5 ng (T1), 10 ng (T2), and 20 ng/mL (T3) on cumulus cell expansion, oocyte maturation, in vitro embryo production, total cell number (TCN) of the blastocyst, and expression of the FGF2 and FGFR2 transcripts in buffalo oocytes and the embryos. Results showed that the effect of FGF2 on the diameter of buffalo COC was significantly higher (P < 0.05) in the T1 group than the other groups at 24h of maturation. The maturation and cleavage rate of oocytes was significantly higher (P < 0.05) in the T3 group than the control, however, the values did not different (P> 0.05) from other groups. The effect of FGF2 on morula and blastocyst yield did not different (P > 0.05) between treatment groups. However, the TCN of the blastocyst was slightly higher (P > 0.05) in the T3 group than the control and other groups. In subsequent trials, the expression of the FGF2 transcript was higher (P < 0.05) in A-grade of oocytes than the C- and D-grade of oocytes, but the expression was not different (P> 0.05) from the B-grade of oocytes. While the FGFR2 expression was higher (P < 0.05) in cumulus cells than any grades of oocytes. The relative abundance of FGF2 and FGFR2 transcripts was significantly higher (P < 0.05) in the 2-cell stage of the embryo than the other stages of embryos. This study was further extended to characterize the FGF2 ligand-binding site in the D3 domain of the buffalo FGF2 receptor. Bioinformatics analysis showed that the bovine FGF2 ligand-binding site in the D3 domain of buffalo was different from the D3 domain of the cattle.

Propagation of goat putative spermatogonial stem cells under growth factors defined serum-free culture conditions.

In the present study, we used a serum-free culture media to propagate goat putative spermatogonial stem cells (SSCs) and evaluated the effect of crucial growth factors on relative expression of some SSC markers and self-renewal related genes. The enriched SSCs were cultured on a homologous Sertoli cell feeder layer in KO-DMEM supplemented with 10% KOSR. Putative SSC colonies emerged between day 6 and 10 which were then characterized by the expression of numerous spermatogonial and pluripotency related markers. After 15 days of subculture, the relative mRNA expression study revealed that 40 ng/mL concentration of Glial cell line-derived neurotrophic factor (GDNF) upregulated the expression of BCL6B, ID4, PLZF, and UCHL1. Moreover, the supplementation of GDNF + bFGF up-regulated the expression of PLZF and BCL6B. UCHL1 expression was higher after addition of GDNF + LIF while, THY1 overexpressed in response to the addition of GDNF + CSF1. These results demonstrated that the goat SSCs were efficiently propagated using a KOSR based serum-free media and the growth factor supplementation markedly influences their gene expression profile.

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

Across farm animal species, the live birth rate obtained with somatic cell nuclear transfer (SCNT) embryos is only <2% compared with >40% obtained with in vitro fertilization (IVF) embryos, primarily due to incomplete nuclear reprogramming which results in aberrant embryonic gene expression. We used RNA sequencing to compare the global transcriptome profile of SCNT and IVF buffalo blastocysts. SCNT blastocysts expressed 17,061 transcripts, of which 941 were unique whereas, IVF blastocysts expressed 17,303 transcripts, of which 1,183 were unique. At ≥2-folds change (p < .05), 331 transcripts were differentially expressed in the two groups among which, 19 were unique, 188 were downregulated and 143 were upregulated in SCNT compared with IVF blastocysts. Many genes affecting pluripotency, trophectoderm development, developmental regulation, and epigenetic modifications were upregulated in SCNT compared with IVF blastocysts. Among the four functional categories analyzed, epigenetic regulators were the most affected. Most of the WNT signaling pathway genes were upregulated whereas, the inhibitors of this pathway, such as DKK1, were downregulated in SCNT blastocysts, suggesting that this pathway is overexpressed in SCNT embryos. Gene Ontology analysis revealed that 25 biological processes, 20 molecular functions, and 24 cellular compartment categories were enriched in SCNT blastocysts. This data can help identify reprogramming errors for improving cloning efficiency.

Selection of Reference miRNAs for Relative Quantification in Buffalo (Bubalus bubalis) Blastocysts Produced by Hand-Made Cloning and In Vitro Fertilization.

Very low birth rate and a high incidence of abnormalities in offspring born from cloned embryos, which have limited the application of cloning technology on a wide scale, are believed to be because of incomplete or aberrant nuclear reprogramming. MicroRNAs (miRNAs) are involved in regulating a wide range of biological processes including reprogramming and embryonic development. Selection of suitable reference miRNAs is critical for normalization of data for accurate relative quantification of miRNAs by quantitative real-time polymerase chain reaction (qRT-PCR), which is currently the most widely used technique for quantifying miRNAs. This study was aimed at identification of reference miRNAs suitable for normalization of qRT-PCR data from blastocyst-stage buffalo embryos produced by handmade cloning and in vitro fertilization (IVF). RNA isolated from cloned and IVF blastocysts was subjected to next-generation sequencing based on which, 12 highly and most consistently expressed miRNAs, which included miR-92a, miR-423, miR-151, Let-7a, miR-103a, miR-93, miR-16b, miR-25, miR-30e, miR-101, miR-127, and miR-197, were selected as candidates for identification of suitable reference miRNAs using three statistical algorithms namely geNorm, NormFinder, and BestKeeper. Based on consensus of the three algorithms, the combination of miRNAs found to be suitable as reference miRNAs were miR-127 and miR-103 for IVF blastocysts; miR-92a and miR-103 for cloned blastocysts, and miR-103, miR-423, and miR-93 across both IVF and cloned blastocysts. The data of this study can be very useful in miRNA expression analysis of blastocyst-stage cloned and IVF embryos.