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.

Exposure to Pulsed Electromagnetic Fields Improves the Developmental Competence and Quality of Somatic Cell Nuclear Transfer Buffalo () 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 ( < 0.05) the relative cell viability and cell proliferation and the expression level of , , , , , and , but decreased ( < 0.

Production of Transgenic Handmade Cloned Goat () 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.

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.

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.

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.

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

Optimization of Serum-Free Culture Conditions for Propagation of Putative Buffalo (Bubalus bubalis) Spermatogonial Stem Cells.

Spermatogonial stem cells (SSCs) self-renew and produce a large number of differentiated germ cells to maintain normal spermatogenesis. However, the growth factors crucial for SSC self-renewal and the mechanism underlying this process remain unclear. In the present study, a serum-free culture media was used to evaluate the effect of several growth factors on the expression of some SSC markers and self-renewal related genes.

Supplementation of Glial Cell Line-Derived Neurotrophic Factor, Fibroblast Growth Factor 2, and Epidermal Growth Factor Promotes Self-Renewal of Putative Buffalo (Bubalus bubalis) Spermatogonial Stem Cells by Upregulating the Expression of miR-20b, miR-21, and miR-106a.

In this study, we investigated the effect of glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor (FGF) 2, and epidermal growth factor (EGF) on the expression of some self-renewal-related microRNAs (miRs) in putative buffalo spermatogonial stem cells (SSCs). The SSCs were cultured on a buffalo Sertoli cell feeder layer, colony formation was observed between 7 and 10 days. The SSC colonies expressed markers specific for undifferentiated type A spermatogonia and pluripotency markers.

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

Inhibition of ERK/MAPK pathway has been shown to decrease DNA methylation via down-regulation of DNA methyltransferases (DNMTs) in several studies suggesting that this pathway plays an important role in regulation of DNA methylation. We examined the relative expression level of seven important genes related to ERK/MAPK pathway and DNMTs (DNMT1, DNMT3a and DNMT3b) by quantitative real-time PCR in buffalo blastocysts produced by Hand-made cloning and compared it with that in blastocyst-stage embryos produced by in vitro fertilization (IVF). The expression level of six of seven genes related to ERK/MAPK pathway examined i.

Establishment, Growth, Proliferation, and Gene Expression of Buffalo (Bubalus bubalis) Transgenic Fetal Fibroblasts Containing Human Insulin Gene, and Production of Embryos by Handmade Cloning Using These Cells.

The aim of the present study was to compare transgenic cells, containing human insulin gene kept under the control of mammary gland-specific buffalo beta-lactoglobulin promoter, and their counterparts, that is, nontransgenic cells, for examining their potential for the production of embryos following somatic cell nuclear transfer (SCNT). The gene construct was delivered into buffalo fetal fibroblasts (BFF) by nucleofection following which, the transfected cells were selected by culture in the presence of G418 for 3 weeks. Transgene integration into BFF genome was confirmed by polymerase chain reaction (PCR) and reverse transcriptase PCR.

Epigenetic Alteration of Donor Cells with Histone Deacetylase Inhibitor m-Carboxycinnamic Acid Bishydroxymide Improves the In Vitro Developmental Competence of Buffalo (Bubalus bubalis) Cloned Embryos.

Epigenetic reprogramming is an indispensable process during the course of mammalian development, but aberrant in cloned embryos. The aim of this study was to examine the effect of donor cell treatment with histone deacetylase (HDAC) inhibitor m-carboxycinnamic acid bishydroxymide (CBHA) on cloned embryo development and establish its optimal concentration. Different concentrations of CBHA (2.

Selection of Suitable Internal Control Genes for Accurate Normalization of Real-Time Quantitative PCR Data of Buffalo (Bubalus bubalis) Blastocysts Produced by SCNT and IVF.

We evaluated the suitability of 10 candidate internal control genes (ICGs), belonging to different functional classes, namely ACTB, EEF1A1, GAPDH, HPRT1, HMBS, RPS15, RPS18, RPS23, SDHA, and UBC for normalizing the real-time quantitative polymerase chain reaction (qPCR) data of blastocyst-stage buffalo embryos produced by hand-made cloning and in vitro fertilization (IVF). Total RNA was isolated from three pools, each of cloned and IVF blastocysts (n = 50/pool) for cDNA synthesis. Two different statistical algorithms geNorm and NormFinder were used for evaluating the stability of these genes.

Retinoic acid induces differentiation of buffalo (Bubalus bubalis) embryonic stem cells into germ cells.

Development of precise and reproducible culture system for in vitro differentiation of embryonic stem (ES) cells into germ cells counts as a major leap forward for understanding not only the remarkable process of gametogenesis, otherwise obscured by limited availability of precursor primordial germ cells (PGCs), but in finally treating the catastrophic infertility. Taking into account the significant role of retinoic acid (RA) during in vivo gametogenesis, we designed the present study to investigate the effects of its stimulation on directing the differentiation of ES cells into germ cells. The effects of RA were analyzed across dose-and-time upon various stages of gametogenesis like PGC induction, meiosis initiation and completion, haploid cell formation and development of the final gamete (oocyte and spermatozoa).

An immunological approach of sperm sexing and different methods for identification of X- and Y-chromosome bearing sperm.

Separation of X- and Y-chromosome bearing sperm has been practiced for selection of desired sex of offspring to increase the profit in livestock industries. At present, fluorescence-activated cell sorter is the only successful method for separation of X- and Y-chromosome bearing sperm. This technology is based on the differences in DNA content between these two types of sperm and has been commercialized for bovine sperm.

Retinoic acid induces differentiation of buffalo (Bubalus bubalis) embryonic stem cells into germ cells.

Development of precise and reproducible culture system for in vitro differentiation of embryonic stem (ES) cells into germ cells counts as a major leap forward for understanding not only the remarkable process of gametogenesis, otherwise obscured by limited availability of precursor primordial germ cells (PGCs), but in finally treating the catastrophic infertility. Taking into account the significant role of retinoic acid (RA) during in vivo gametogenesis, we designed the present study to investigate the effects of its stimulation on directing the differentiation of ES cells into germ cells. The effects of RA were analyzed across dose-and-time upon various stages of gametogenesis like PGC induction, meiosis initiation and completion, haploid cell formation and development of the final gamete (oocyte and spermatozoa).

Treatment of Donor Cells and Reconstructed Embryos with a Combination of Trichostatin-A and 5-aza-2'-Deoxycytidine Improves the Developmental Competence and Quality of Buffalo Embryos Produced by Handmade Cloning and Alters Their Epigenetic Status and Gene Expression.

The application of cloning technology on a large scale is limited by very low offspring rate primarily due to aberrant or incomplete epigenetic reprogramming. Trichostatin A (TSA), a histone deacetylase inhibitor, and 5-aza-2'-deoxycytidine (5-aza-dC), an inhibitor of DNA methyltransferases, are widely used for altering the epigenetic status of cloned embryos. We optimized the doses of these epigenetic modifiers for production of buffalo embryos by handmade cloning and examined whether combined treatment with these epigenetic modifiers offered any advantage over treatment with the individual epigenetic modifier.

Valproic Acid Increases Histone Acetylation and Alters Gene Expression in the Donor Cells But Does Not Improve the In Vitro Developmental Competence of Buffalo (Bubalus bubalis) Embryos Produced by Hand-Made Cloning.

Use of histone deacetylase inhibitors (HDACis) is believed to improve the developmental competence and quality of cloned embryos produced. We examined the effects of treatment of buffalo fibroblasts with valproic acid (VPA), a HDACi on these cells and on embryos produced from them by hand-made cloning. VPA treatment (1.

Activation and Inhibition of The Wnt3A Signaling Pathway in Buffalo (Bubalus bubalis) Embryonic Stem Cells: Effects of WNT3A, Bio and Dkk1.

Background: This research studies the effects of activation and inhibition of Wnt3A signaling pathway in buffalo (Bubalus bubalis) embryonic stem (ES) cell-like cells.

Materials And Methods: To carry on this experimental study, the effects of activation and inhibition of Wnt3A signaling in buffalo ES cell-like cells were examined using Bio (0.5 mM) combined with WNT3A (200 ng/ml), as an activator, and Dickkopf-1 (Dkk1, 250 ng/ml), as an inhibitor, of the pathway.

Cumulus cell-conditioned medium supports embryonic stem cell differentiation to germ cell-like cells.

Cumulus cells provide cellular interactions and growth factors required for oogenesis. In vitro studies of oogenesis are limited primarily because of the paucity of their source, first trimester fetal gonads, and the small number of germ lineage precursor cells present within these tissues. In order to understand this obscure but vitally important process, the present study was designed to direct differentiation of embryonic stem (ES) cells into germ lineage cells.

RETRACTED: Bone morphogenetic protein 4 (BMP4) induces buffalo (Bubalus bubalis) embryonic stem cell differentiation into germ cells.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy).

Optimization of Buffalo (Bubalus bubalis) Embryonic Stem Cell Culture System.

Objective: In order to retain an undifferentiated pluripotent state, embryonic stem (ES) cells have to be cultured on feeder cell layers. However, use of feeder layers limits stem cell research, since experimental data may result from a combined ES cell and feeder cell response to various stimuli.

Materials And Methods: In this experimental study, a buffalo ES cell line was established from in vitro derived blastocysts and characterized by the Alkaline phosphatase (AP) and immunoflourescence staining of various pluripotency markers.

Development, Characterization, and Pluripotency Analysis of Buffalo (Bubalus bubalis) Embryonic Stem Cell Lines Derived from In Vitro-Fertilized, Hand-Guided Cloned, and Parthenogenetic Embryos.

We present the derivation, characterization, and pluripotency analysis of three buffalo embryonic stem cell (buESC) lines, from in vitro-fertilized, somatic cell nuclear-transferred, and parthenogenetic blastocysts. These cell lines were developed for later differentiation into germ lineage cells and elucidation of the signaling pathways involved. The cell lines were established from inner cell masses (ICMs) that were isolated manually from the in vitro-produced blastocysts.

Molecular characterization and expression of buffalo (Bubalus bubalis) DEAD-box family VASA gene and mRNA transcript variants isolated from testis tissue.

VASA is a member of the DEAD-box protein family that plays an indispensable role in mammalian spermatogenesis, particularly during meiosis. In the present study, we isolated, sequenced, and characterized VASA gene in buffalo testis. Here, we demonstrated that VASA mRNA is expressed as multiple isoforms and uses four alternative transcriptional start sites (TSSs) and four different polyadenylation sites.

Establishment of Trophectoderm Cell Lines from Buffalo (Bubalus bubalis) Embryos of Different Sources and Examination of In Vitro Developmental Competence, Quality, Epigenetic Status and Gene Expression in Cloned Embryos Derived from Them.

Despite being successfully used to produce live offspring in many species, somatic cell nuclear transfer (NT) has had a limited applicability due to very low (>1%) live birth rate because of a high incidence of pregnancy failure, which is mainly due to placental dysfunction. Since this may be due to abnormalities in the trophectoderm (TE) cell lineage, TE cells can be a model to understand the placental growth disorders seen after NT. We isolated and characterized buffalo TE cells from blastocysts produced by in vitro fertilization (TE-IVF) and Hand-made cloning (TE-HMC), and compared their growth characteristics and gene expression, and developed a feeder-free culture system for their long-term culture.