Reduced sperm number of murrah (Bubalus bubalis) bull semen in french mini-straw affects kinetic and functional competence after cryopreservation.

BACKGROUND: Extensive dilution of cattle semen with tris-based extender compromises certain sperm kinetic and functional traits following cryopreservation. OBJECTIVE: To study sperm functions of buffalo bulls under high dilution rates. MATERIALS AND METHODS: Twenty-four ejaculates were harvested twice a week from four buffalo bulls, and diluted to sperm concentrations of 80, 60, 40 and 20 million/mL. Diluted samples were filled in straws, equilibrated at refrigeration temperature for 4 h, and frozen in liquid nitrogen. Frozen sperm samples were thawed for evaluation of kinetic and functional attributes. RESULTS: Compared to 20 million/mL (million/mL) sperm sample, the total motility, progressive motility and rapid motility were reduced (P < 0.05) in 5 million/mL sample. The proportion of live sperm were significantly (P < 0.05) higher in 10, 15 and 20 million/mL samples than in 5 million/mL sample. The percentage of moribund sperm, dead sperm, and sperm with lipid per oxidation increased significantly (P < 0.05) in 5 million/mL sample. CONCLUSION: The reduction of sperm concentrations to < 10 million/mL affects post-thaw Buffalo sperm kinetic and functional attributes.. https://doi.org/10.54680/fr24110110712.

Genome-Wide DNA Methylation and Its Effect on Gene Expression During Subclinical Mastitis in Water Buffalo.

Subclinical mastitis (SCM) in buffalo is one of the most challenging paradoxes for the dairy sector with very significant milk production losses and poses an imminent danger to milch animal's milk-producing ability. We present here the genome-wide methylation specific to SCM in water buffalo and its consequential effect on the gene expression landscape for the first time. Whole-genome DNA methylation profiles from peripheral blood lymphocytes and gene expression profiles from milk somatic cells of healthy and SCM cases were catalogued from the MeDIP-Seq and RNA-Seq data. The average methylation in healthy buffaloes was found to be higher than that in the SCM-infected buffaloes. DNA methylation was abundant in the intergenic region followed by the intronic region in both healthy control and SCM groups. A total of 3,950 differentially methylated regions (DMRs) were identified and annotated to 370 differentially methylated genes (DMGs), most of which were enriched in the promoter region. Several important pathways were activated due to hypomethylation and belonged to the Staphylococcus aureus infection, Th17 cell differentiation, and antigen processing and presentation pathways along with others of defense responses. DNA methylome was compared with transcriptome to understand the regulatory role of DNA methylation on gene expression specific to SCM in buffaloes. A total of 4,778 significant differentially expressed genes (DEGs) were extracted in response to SCM, out of which 67 DMGs were also found to be differentially expressed, suggesting that during SCM, DNA methylation could be one of the epigenetic regulatory mechanisms of gene expression. Genes like CSF2RB, LOC102408349, C3 and PZP like, and CPAMD8 were found to be downregulated in our study, which are known to be involved in the immune response to SCM. Association of DNA methylation with transposable elements, miRNAs, and lncRNAs was also studied. The present study reports a buffalo SCM web resource (BSCM2TDb) available at http://webtom.cabgrid.res.in/BSCM2TDb that catalogues all the mastitis-related information of the analyses results of this study in a single place. This will be of immense use to buffalo researchers to understand the host-pathogen interaction involving SCM, which is required in endeavors of mastitis control and management.

Establishment of Repertoire of Placentome-Associated MicroRNAs and Their Appearance in Blood Plasma Could Identify Early Establishment of Pregnancy in Buffalo (Bubalus bubalis).

Precise early pregnancy diagnosis in dairy animals is of utmost importance for an efficient dairy production system. Not detecting a dairy animal pregnant sufficiently early after the breeding results to extending the unproductive time of their milk production cycle and causes substantial economic loss for a dairy producer. At present, the most conventional and authentic pregnancy confirmation practice in cows and buffaloes is rectal palpation of the reproductive organs at Days 35-40 after insemination, which sometime leads to considering an animal as false pregnant. Other alternative methods available for early pregnancy diagnosis lack either accuracy or reproducibility or require elaborate instrumentation and laboratory setup not feasible to practice at farmers' doorstep. The present study was aimed at establishment of the microRNA (miRNA) repertoire of the placentome in buffaloes, which could capture the event of the cross talk between a growing embryo and a dam, through fetal cotyledons and maternal caruncles, and thus could hint at the early pregnancy establishment event in ruminants. Total RNA was isolated from buffalo placentome tissues during early stages of pregnancy (at Day < 25 and Days 30-35), and global small RNA analysis was performed by using Illumina single-end read chemistry and Bubalus bubalis genome. A total of 2,199 miRNAs comprising 1,620 conserved and 579 non-conserved miRNAs were identified. Stringent functional miRNA selection criteria could predict 20 miRNAs worth evaluating for their abundance in the plasma of pregnant, non-pregnant, cyclic non-bred, and non-cyclic prepubertal animals. Eight of them (viz., miR-195-5p, miR-708-3p, miR-379-5p, miR-XX1, miR-XX2, miR-130a-3p, miR-200a-3p, and miR-27) displayed typical abundance patterns in the plasma samples of the animals on Day 19 as well as Day 25 post-insemination, thus making them ambiguous candidates for early pregnancy detection. Similarly, higher abundance of miR-200a-3p and miR130a-3p in non-pregnant animals was indicative of their utility for detecting the animals as not pregnant. Most interestingly, miR-XX1 and miR-XX2 were very characteristically abundant only in pregnant animals. In silico target prediction analysis confirmed that these two miRNAs are important regulators of cyclooxygenase-2 (COX-2) and cell adhesion molecule-2 (CADM-2), both of which play a significant role in the implantation process during feto-maternal cross talk. We interpret that circulatory miR-XX1 and miR-XX2 in blood plasma could be the potential biomarkers for early pregnancy detection in buffaloes.

Unusual interplay of contrasting selective pressures on ß-defensin genes implicated in male fertility of the Buffalo (Bubalus bubalis).

BACKGROUND: The buffalo, despite its superior milk-producing ability, suffers from reproductive limitations that constrain its lifetime productivity. Male sub-fertility, manifested as low conception rates (CRs), is a major concern in buffaloes. The epididymal sperm surface-binding proteins which participate in the sperm surface remodelling (SSR) events affect the survival and performance of the spermatozoa in the female reproductive tract (FRT). A mutation in an epididymal secreted protein, beta-defensin 126 (DEFB-126/BD-126), a class-A beta-defensin (CA-BD), resulted in decreased CRs in human cohorts across the globe. To better understand the role of CA-BDs in buffalo reproduction, this study aimed to identify the BD genes for characterization of the selection pressure(s) acting on them, and to identify the most abundant CA-BD transcript in the buffalo male reproductive tract (MRT) for predicting its reproductive functional significance. RESULTS: Despite the low protein sequence homology with their orthologs, the CA-BDs have maintained the molecular framework and the structural core vital to their biological functions. Their coding-sequences in ruminants revealed evidence of pervasive purifying and episodic diversifying selection pressures. The buffalo CA-BD genes were expressed in the major reproductive and non-reproductive tissues exhibiting spatial variations. The Buffalo BD-129 (BuBD-129) was the most abundant and the longest CA-BD in the distal-MRT segments and was predicted to be heavily O-glycosylated. CONCLUSIONS: The maintenance of the structural core, despite the sequence divergence, indicated the conservation of the molecular functions of the CA-BDs. The expression of the buffalo CA-BDs in both the distal-MRT segments and non-reproductive tissues indicate the retention the primordial microbicidal activity, which was also predicted by in silico sequence analyses. However, the observed spatial variations in their expression across the MRT hint at their region-specific roles. Their comparison across mammalian species revealed a pattern in which the various CA-BDs appeared to follow dissimilar evolutionary paths. This pattern appears to maintain only the highly efficacious CA-BD alleles and diversify their functional repertoire in the ruminants. Our preliminary results and analyses indicated that BuBD-129 could be the functional ortholog of the primate DEFB-126. Further studies are warranted to assess its molecular functions to elucidate its role in immunity, reproduction and fertility.

Propensity in low-grade oocytes for delayed germinal vesicle breakdown compromises the developmental ability of sub-optimal grade Bubalus bubalis oocytes.

SummaryMaturing oocytes have diverse developmental potential and good quality oocytes exhibit a better ability to attain physiological milestones in a time-dependent manner. This situation necessitates the confirmation of oocyte developmental status more precisely under an in vitro embryo production (IVEP) regime. The aim of this study was to explain timely events in germinal vesicle breakdown (GVBD), an important milestone of oocyte nuclear maturation, to delineate the developmental capacity of Bubalus bubalis oocytes. In addition, the expression profile of genes responsible for GVBD was assessed in order to understand the molecular context responsible for GVBD. The chronology of GVBD events at different time intervals during in vitro maturation (IVM) suggests that the rate at which oocytes undergo GVBD was strikingly different in the brilliant cresyl blue (BCB)+ and BCB- groups. The expression of AKT and CDC25B genes for BCB+ oocytes was maximum at 8 h of IVM, and CCNB (cyclin B) peaked at around 10 h, which suggested that GVBD was finished after 10 h in BCB+ oocytes, whereas the expression of AKT and CDC25B was found to peak at around 12-14 h of IVM. This difference consequently delays the GVBD event by 2-4 h in BCB- oocytes. Poor abundance of gene transcripts was mainly implicated in delay and lower rate of GVBD in BCB- oocytes which in turn strongly affected the translational ability of oocytes to blastocysts. The findings of this study support the idea that there is a propensity in sub-optimal grade oocytes for delayed GVBD that compromises the developmental ability of low grade buffalo oocytes. The study highlights the very small, but importantly vital and separate, time window of the GVBD event during which the competence levels of buffalo oocytes are altered along with their translational ability to develop into the prospective embryos.

Identification of biomarker candidates for fertility in spermatozoa of crossbred bulls through comparative proteomics.

Associations between expression of some proteins in spermatozoa and fertility have been sought in recent years to identify the male fertility markers. Since the incidence of sub-fertility is high in crossbred bulls, the present investigation was carried out on high- and low-fertile crossbred bulls to identify fertility markers in spermatozoa through proteomics approach. Sperm proteome of high-fertile bulls were compared with low-fertile bulls using 2D-DIGE and MALDI-TOF-MS techniques and the results were validated with immuno-blotting. The proteins MDH2, ENO1, RIBC1, CAPN7, ATP5D, LacA like protein-2 like, NCAPD3, DECR1, GCNT2, GDI2, TOP and USP12 were over expressed in high-fertile spermatozoa, whereas DST like isoform 1, TMEM43 and BSP1 were over expressed in low-fertile spermatozoa (P < 0.05). The differential expression ranged from 1.57 (GDI2) to 5.1 (BSP1) fold between the two groups. Based on the GO annotation, majority of them were involved in cellular and metabolic processes, with catalytic and binding activities, and localized in cell and organelles. Among these proteins, ENO1 and BSP1 were selected based on the degree of differential expression and reliability in identification, for further validation. Immuno-blotting studies indicated that ENO1expression was positively correlated (P < 0.05) while the expression of BSP1 was negatively (P < 0.01) correlated with bull fertility. The proportion of capacitated spermatozoa in frozen thawed spermatozoa of low-fertile bulls was higher (P < 0.05) as compared to high-fertile bulls. Collectively, the study identified some potential molecules in spermatozoa of bulls, which may act as a panel of biomarkers for fertility.

Development of an in vitro oviduct epithelial explants model for studying sperm-oviduct binding in the buffalo.

In this study, we developed an in vitro model for studying sperm-oviduct binding in the buffalo. Oviduct explants were prepared by overnight culture of epithelial cells in TCM-199 medium under 5% CO2 at 38.5 °C. Cryopreserved spermatozoa from buffalo bulls (n = 4) were incubated with the oviduct explants, and the sperm-oviduct explants complex was stained with JC-1. The effect of sperm concentration (2, 3 and 4 million), size of the oviduct explants (<0.2, 0.2-0.3, 0.3-0.4 and >0.4 mm2 ) and time of incubation (1 hr and 4 hr) on binding index (BI-number of sperm bound to unit area of explants) was studied. No significant difference was observed in the BI among <0.2, 0.2-0.3 and 0.3-0.4 mm2 size of explants; however, the BI decreased significantly (p < .05) when the size of explants exceeded 0.4 mm2 . The BI decreased significantly (p < .05) when the sperm concentration was increased to 4 million, while the duration of incubation did not have any significant effect on the BI. The interaction of bulls with explants size, sperm concentration and incubation time was not significant. The developed assay has the potential to be used as an in vitro model for studying sperm-oviduct binding in the buffalo.

Evolutionary dynamics of meiotic recombination hotspots regulator PRDM9 in bovids.

Hybrid sterility or reproductive isolation in mammals has been attributed to allelic incompatibilities in a DNA-binding protein PRDM9. Not only is PRDM9 exceptional in being the only known 'speciation gene' in vertebrates, but it is also considered to be the fastest evolving gene in the genome. The terminal zinc finger (ZF) domain of PRDM9 specifies genome-wide meiotic recombination hotspot locations in mammals. Intriguingly, PRDM9 ZF domain is highly variable between as well as within species, possibly activating different recombination hotspots. The present study characterized the full-length coding sequence of PRDM9 in cattle and buffalo and explored the diversity of the ZF array in 514 samples from different bovids (cattle, yak, mithun, and buffalo). Substantial numerical and sequence variability were observed in the ZFs, with the number of repeats ranging from 6 to 9 in different bovines. Sequence analysis revealed the presence of 37 different ZFs in cattle, 3 in mithun, 4 in yak, and 13 in buffaloes producing 41 unique PRDM9 alleles in these species. The posterior mean of dN/dS or omega values calculated using Codeml tool of PAMLX identified sites -5, -1, +2, +3, +4, +5, and +6 in the ZF domain to be evolving positively in the studied species. Concerted evolution which typifies the evolution of this gene was consistently evident in all bovines. Our results demonstrate the extraordinary diversity of PRDM9 ZF array across bovines, reinforcing similar observations in other metazoans. The high variability is suggestive of unique repertoire of meiotic recombination hotspots in each species.

Identification of suitable combinations of in vitro sperm-function test for the prediction of fertility in buffalo bull.

The present study assessed sperm functional characteristics in the frozen-thawed semen of buffalo bulls and estimated their relationship with field fertility. Frozen semen samples from three different freezing operations each from nine Murrah buffalo bulls were used for the assessment of different sperm functions related to fertilizing potential. Bulls were classified into high (n = 2), medium (n = 5), and low (n = 2) fertile based on adjusted field fertility. The sperm functions estimated included membrane integrity using carboxyfluorescein diacetate-propidium iodide, acrosome reaction status using fluorescein isothiocyanate peanut agglutinine, status of apoptosis using Annexin-V, protamine deficiency using Chromomycin A3, membrane stability using Merocyanine 540 and lipid peroxidation status using 4, 4-difluoro-4-bora-3a, 4a-diaza-s-indacene. The relationship between the proportion of live acrosome-intact spermatozoa and fertility was positive and significant (r = 0.59; P = 0.001). The proportion of moribund spermatozoa showed a significantly negative correlation with fertility (r = -0.50; P = 0.008). Similarly, the relationship of spermatozoa with unstable membrane (r = -0.51; P = 0.007), necrotic (r = - 0.42; P = 0.028), early necrotic (r = -0.42; P = 0.031), and apoptotic spermatozoa (r = -0.39; P = 0.046) with bull fertility was negative and significant. The correlation between the protamine-deficient spermatozoa and fertility was negative, but not significant. Among different combinations of tests, live acrosome-intact spermatozoa and lipid peroxidation status of spermatozoa revealed high positive correlation with buffalo bull fertility (adjusted R2 = 0.73, C[p] = 0.80). These preliminary findings may help in developing tools for assessing fertility of buffalo bulls, once validated in more animals.

Follicle-stimulating hormone-induced rescue of cumulus cell apoptosis and enhanced development ability of buffalo oocytes.

The effect of follicle-stimulating hormone (FSH) on apoptotic status of cumulus cells, expression of proapoptotic and antiapoptotic genes, and development rate of in vitro fertilization-produced buffalo embryos were investigated. FSH supplementation in in vitro maturation-medium resulted in a dose-dependent reduction in the expression of proapoptotic genes namely, BCL2-associated X protein (BAX), cytochrome c, and caspase-3 and increase in the expression of antiapoptotic genes such as B-cell lymphoma 2 (BCL2) and X-linked inhibitor of apoptosis protein (XIAP) in cumulus cells of mature oocyte. Cumulus expansion, oocyte maturation, cleavage, and blastocyst development rates were significantly higher (P < 0.05) in 5 and 10-µg/mL FSH-supplemented groups as compared with control. Significant increase in the expression of FSH receptor messenger RNA was also found with 5 and 10-µg/mL FSH (P < 0.05). Terminal deoxynucleotidyl transferase dUTP nick end labeling assay confirmed that the population of apoptotic cumulus cells of matured oocytes was reduced in the FSH-treated groups as compared with control (P < 0.05). In conclusion, our data suggest that FSH may attenuate apoptosis in cumulus cells via mitochondria-dependent apoptotic pathway by increasing XIAP expression, resulting in a more favorable ratio of BCL2/BAX expression and decreasing the cytochrome c and caspase-3 expression, eventually contributing to developmental competence of oocytes. The information generated will help in improving the in vitro embryo production program in buffalo.

Splice variants and seasonal expression of buffalo HSF genes.

In eukaryotes, the heat shock factors (HSFs) are recognized as the master regulator of the heat shock response. In this respect, the genes encoding the heat shock factors seem to be important for adaptation to thermal stress in organisms. Despite this, only few mammalian HSFs has been characterized. In this study, four major heat shock factor genes viz. HSF-1, 2, 4, and 5 were studied. The main objective of the present study was to characterize the cDNA encoding using conserved gene specific primers and to investigate the expression status of these buffalo HSF genes. Our RT-PCR analysis uncovered two distinct variants of buffalo HSF-1 and HSF-2 gene transcripts. In addition, we identified a variant of the HSF5 transcript in buffalo lacking a DNA-binding domain. In silico analysis of deduced amino acid sequences for buffalo HSF genes showed domain architecture similar to other mammalian species. Changes in the gene expression profile were noted by quantitative real-time PCR (qRT-PCR) analysis. We detected the transcript of buffalo HSF genes in different tissues. We also evaluated the seasonal changes in the expression of HSF genes. Interestingly, the transcript level of HSF-1 gene was found upregulated in months of high and low ambient temperatures. In contrast, the expression of the HSF-4 and 5 genes was found to be downregulated in months of high ambient temperature. This suggests that the intricate balance of different HSFs is adjusted to minimize the effect of seasonal changes in environmental conditions. These findings advance our understanding of the complex, context-dependent regulation of HSF gene expression under normal and stressful conditions.

Comparative expression profiling of insulin-like growth factor binding protein-5 in milk of Bos indicus and Bubalus bubalis during lactation.

Insulin-like growth factor binding protein-5 (IGFBP-5) is a key molecule in mammary gland development, which facilitates the removal of mammary epithelial cells (MECs) by apoptosis that takes place during remodeling of the mammary gland during involution. IGFBP-5 binds with IGFs for their bioavailability. IGFBP-5 has been reported to perform pleiotropic roles such as cellular apoptosis, proliferation and differentiation. To understand the role of IGFBP-5 during lactation and clinical mastitis, expression profiling of IGFBP-5 at the protein level was performed in both indigenous cows (Bos indicus) and buffaloes (Bubalus bubalis) belonging to two different breeds - Sahiwal cows and Murrah buffaloes. Reverse-transcriptase PCR (RT-PCR) of IGFBP-5 mRNA confirmed its expression in milk somatic cells and MECs of Sahiwal cows. ELISA was performed for quantitative measurement of IGFBP-5 concentrations in milk during different days (0, 50, 100, 150, 200, 250 and 300) of lactation, during the involution period and in animals exhibiting short lactation and clinical mastitis. The highest concentration of IGFBP-5 in milk was observed during the involution period followed by colostrum, late and early lactation, respectively, in both cattle and buffaloes. No significant difference in the concentration of IGFBP-5 was observed during the first 150 days of lactation between cows and buffaloes. However, higher concentration of IGFBP-5 was observed in cows during late lactation (200 to 300 days) in comparison with buffaloes. To validate the ELISA data, quantitative real-time PCR was performed in MECs of Sahiwal cows. The relative mRNA abundance of IGFBP-5 was found to be significantly (P<0.05) higher on day 15 than between 50 and 150 days of lactation in case of Sahiwal cows. Highest mRNA expression of IGFBP-5 was observed around 300 days of lactation followed by 200 and 250 days (P<0.05), respectively. Murrah buffaloes showed low levels of IGFBP-5 protein in milk as compared with Sahiwal cows during lactation in ELISA. Animals having history of short lactation length (short lactating animals) showed higher levels of IGFBP-5 expression (at protein level) in comparison with normal lactating animals. We propose that higher level IGFBP-5 expression may have functional significance in lactation persistency. As a pro-apoptotic molecule, higher expression of IGFBP-5 was observed to be inversely related to lactation length and milk production.

Characterization of oocyte-expressed GDF9 gene in buffalo and mapping of its TSS and putative regulatory elements.

Summary In spite of emerging evidence about the vital role of GDF9 in determination of oocyte competence, there is insufficient information about its regulation of oocyte-specific expression, particularly in livestock animals. Because of the distinct prominence of buffalo as a dairy animal, the present study was undertaken to isolate and characterize GDF9 cDNA using orthologous primers based on the bovine GDF9 sequence. GDF9 transcripts were found to be expressed in oocytes irrespective of their follicular origin, and shared a single transcription start site (TSS) at -57 base pairs (bp) upstream of ATG. Assignment of the TSS is consistent with the presence of a TATA element at -23 of the TSS mapped in this study. Localization of a buffalo-specific minimal promoter within 320 bp upstream of ATG was consolidated by identification of an E-box element at -113bp. Presence of putative transcription factor binding sites and other cis regulatory elements were analyzed at ~5 kb upstream of TSS. Various germ cell-specific cis-acting regulatory elements (BNCF, BRNF, NR2F, SORY, Foxh1, OCT1, LHXF etc.) have been identified in the 5' flanking region of the buffalo GDF9 gene, including NOBOX DNA binding elements and consensuses E-boxes (CANNTG). Presence of two conserved E-boxes found on buffalo sequence at -520 and -718 positions deserves attention in view of its sequence deviation from other species. Two NOBOX binding elements (NBE) were detected at the -3471 and -203 positions. The fall of the NBE within the putative minimal promoter territory of buffalo GDF9 and its unique non-core binding sequence could have a possible role in the control of the core promoter activity.

Identification of some unknown transcripts from SSH cDNA library of buffalo follicular oocytes.

A buffalo oocyte-specific subtracted cDNA library was constructed to identify exclusively or preferentially oocyte-expressed genes. The library represented an enriched population of transcripts obtained from oocytes of diverse ovarian follicular origin and at different stages of in vitro maturation. A total of 1173 high-quality sequences of oocyte-specific genes were clustered into 645 unique sequences, out of which 65.76% were represented as singlets and 34.26% as contig expressed sequence tags (ESTs; clusters). Analysis of sequences revealed that 498 of these sequences were identified as a known sequence in mammalian species including buffalo, 103 as uncharacterized ESTs and 44 unknown sequences including 1 novel EST, so far not reported in any species. Gene ontology annotation classified these sequences into functional categories of cellular events and biological processes associated with oocyte competence. Expression status of the isolated unknown ESTs confirmed that many of these are expressed in oocytes exclusively and in others preferentially, some in excess of 80-fold greater in comparison with a variety of somatic tissues. The isolated novel EST was detected to be expressed exclusively in oocytes and testicular cells only. To our knowledge, this is the first report giving a detailed transcriptome account of oocyte-expressed genes in buffalo. This study will provide important information on the physiological control of oocyte development, as well as many questions yet to be addressed on the reproductive process of buffalo.

Kinetics of GDF9 expression in buffalo oocytes during in vitro maturation and their associated development ability.

The capacity of fully grown oocytes to regulate their own microenvironment by secreted paracrine factors contribute to their developmental competence. In spite of growing evidence about the vital role of Growth Differentiation Factor 9 (GDF9) in determination of oocyte developmental competence, there is insufficient information about time dependent behavior of its expression during in vitro maturation (IVM) to have definite understanding about at what time point during IVM it plays most crucial role. The study reports the kinetics of GDF9 expression under four different IVM supplement conditions in buffalo oocytes and their concomitant development rate up to blastocyst. Oocytes matured under an ideal media condition with all supplements and those cultured with only FSH resulted in significantly higher cumulus expansion, nuclear maturation, cleavage and blastocyst rates. GDF9 expression at both mRNA and protein levels at different time points of IVM revealed that magnitude of mRNA abundance at 8h of IVM was most important towards imparting development competence to buffalo oocytes. Appearance of GDF9 protein in maturing oocytes was found asynchronous with mRNA appearance in the time course of IVM suggesting possible posttranscriptional regulation of this gene under dynamic oocyte cumulus cell communication process. Abundance of mature GDF9 protein at 16 h was most consistently related with all oocyte development parameters.

Embryonic genome activation events in buffalo (Bubalus bubalis) preimplantation embryos.

Embryonic genome activation (EGA) is the first major step towards successful initiation of preimplantation development, which culminates in the formation of implantation-competent embryos. EGA occurs at species-specific embryonic cell stages. In the present work, EGA was identified for buffalo embryos by studying the development rate of embryos in normal as well as imposed transcription block conditions, analyzing bromo-uridine triphosphate (BrUTP) incorporation rates as evidence of de novo transcription initiation, and studying the expression status of eukaryotic translation initiation factor 1A (eIF1A), U2 auxiliary splicing factor (U2AF), and polyadenylate polymerase (PAP) genes at different embryonic cell stages. Under normal, in vitro fertilization and culture conditions, about 26% and 17% of oocytes could reach morula and blastocyst stages, respectively, but no embryos could progress beyond 8-cell stages in presence of α-amanitin. Culturing embryos in the presence of BrUTP revealed a marked increase in its incorporation between 4- and 8-cell stages. All genes studied displayed an abrupt increase in expression between 4- and 8-cell stages; PAP expression was upregulated earlier from 2- to 4-cell stages. About 65% of PAP transcripts from the 4-cell stage and more than 70% of eIF1A, U2AF, and PAP transcripts at 8-cell stage embryos were found to be synthesized de novo. Together, these data suggest that a minor EGA in buffalo embryos happens from 2- to 4-cell stages, while the major EGA takes place from 4- to 8-cell stage transition.

Effect of varying glucose concentrations during in vitro maturation and embryo culture on efficiency of in vitro embryo production in buffalo.

This study was aimed to optimize glucose level at different stages of buffalo in vitro embryo production procedure. Three glucose levels (1.5, 5.6 and 10 mm) along with a control (0 mm) were used at three phases of in vitro fertilisation (IVF) procedure viz. in vitro maturation (IVM), in vitro culture (IVC-I) (12-72 hpi) and IVC-II (72 hpi to 7 dpi). Maturation rate of oocytes was found different under different glucose concentrations, and significantly more number of oocytes reached to MII under 5.6 mm glucose. The glucose levels at each phase (IVM, IVC-I and IVC-II) individually had significant effect on blastocyst rate, and the level used at one phase had significant effect on the outcome of next phase. Complete withdrawal of glucose from any of these stages irrespective of concentrations used at subsequent stage/s resulted in significantly lower number of blastocysts. However, the changing levels of glucose had differential effects during different phases of IVF steps. The most prominent effect of glucose level was observed during IVM. The presence of 5.6 mm glucose at all stages was most effective to yield highest blastocyst rate in buffalo IVF system.

Characterization of serum-free buffalo granulosa cell culture and analysis of genes involved in terminal differentiation from FSH- to LH-responsive phenotype.

In the present study, buffalo granulosa cells were cultured under serum-free conditions and characterized to study the changes in gene expression associated with the transition of granulosa cells from estrogen- to progesterone-secreting phenotype. The cells were cultured in vitro under completely serum-free conditions for 8 d. Gene expression and hormone analysis showed that on day 4 granulosa cells exhibit FSH responsiveness with preovulatory phenotype having highest CYP19 gene expression and 17ß-estradiol production, whereas a significant increase in transcript abundance of STAR, CYP11, and HSD3B genes accompanied with an increase in progesterone production was observed on day 8. Cells treated with LH on day 4 followed by gene expression analysis at 1, 2, 4, 6, 18, and 24 h showed significant increase in transcripts of LH-responsive genes. In conclusion, culture condition used in the present study showed that granulosa cells were FSH responsive and attained attributes of granulosa cells of dominant follicles at day 4 with highest CYP19 and LHR gene expression beyond which they acquired the ability to luteinize and thus were more LH responsive. In addition, after LH treatment, analysis of early LH-responsive genes (EGR2, RUNX1, and NR4A1) on day 4 showed that granulosa cells at this stage in culture exhibits phenotype similar to that of preovulatory follicles before LH surge in vivo and corresponds to the in vivo transition of well-orchestrated gene expression profile after LH surge. The characterized culture conditions represent a suitable in vitro model for analysis of genes involved in terminal differentiation of granulosa cells from FSH- to LH-responsive phenotype during folliculogenesis in buffalo.

A novel method of plasmid isolation using laundry detergent.

Since the discovery of plasmid, various methods have been developed to isolate plasmid DNA. All the methods have one common and important target of isolating plasmid DNA of high quality and quantity in less time. These methods are not completely safe because of use of toxic chemicals compounds. The developed protocol for plasmid extraction is based on the alkaline lysis method of plasmid preparation (extraction atpH 8.0) with slight modifications. Cell lysis reagent sodium dodecyl sulfate is replaced by lipase enzyme present in laundry detergent. A good plasmid preparation can be made, which is well suited for subsequent molecular biology applications. By taking safety measures on count, contaminants like, RNA and protein can be completely avoided with maximized plasmid yield. The resultant plasmid quality and quantity can be well comparable to other prevalent methods.

Development of a competitive quantitative PCR strategy for evaluating the expression stability of 18s rRNA during in vitro maturation of buffalo (Bubalus bubalis) follicular oocytes.

The present work describes the development of a quantitative competitive PCR strategy for quantifying the relative abundance of 18s rRNA transcripts in buffalo oocytes during in vitro maturation (IVM). As a method, the competitive PCR overcomes some of the shortcomings of conventional reverse transcriptase polymerase chain reaction (RT-PCR) procedure making it a more authentic quantitative method. A composite primer based approach was used to generate the competitor cDNA to be used as external control. Validity of the method for its efficiency was demonstrated by quantitative analysis of the competition parameters. Using this method the relative abundance of buffalo oocyte 18s rRNA transcript over the period of IVM was found to vary within a narrow range of 0.93-1.06 folds which establishes the accuracy of the method and reflects the stability of its expression during IVM. This qualifies the use of this house keeping gene as a valid internal control in studies investigating the gene expression pattern in buffalo oocytes. The competitive PCR approach described in this study could be used for quantification of other transcripts from a limited number of oocytes where a conventional RT-PCR method is either difficult to use or multiplexing it with highly abundant house keeping genes is apparently problematic.