Simarjeet Kaur

Subfertility Problems Leading to Disposal of Breeding Bulls

Subfertility problems are encountered frequently in the cattle and buffalo bulls commercially maintained for semen production in dairy farms and under field conditions for natural insemination. Reports are scarce on the incidence of subfertility in breeding bulls, especially in India. The objective of the present study was to assess the incidence of the male reproductive anomalies leading to disposal of bovine bulls at GADVASU dairy farm, Ludhiana, Punjab (India). Data on frequency of various subfertility and disposal pattern of bulls maintained at the dairy farm, GADVASU, were collected for 12 yrs (1999 to 2010) and compiled from different record registers. Percentage of bulls that produced freezable semen (out of reserved ones) was less in cattle (25.641%) as compared to that of buffalo (30.4%). Various subfertility traits like poor libido and unacceptable seminal profile were found to be the significant reasons (p<0.01) for culling of the breeding bulls. Inadequate sex drive and poor semen quality were the main contributing factors for bull disposal in cattle whereas poor semen freezability was most frequently observed in buffalo bulls. All the male reproductive traits were significantly different (p<0.05) for the periods of birth, except for semen volume, initial motility (IM), age at last semen collection (ALSC) and age at disposal. The ages at first and last semen collection as well as freezing (i.e. AFSC, ALSC and AFSF, ALSF, respectively) and age at disposal (AD) were higher in buffalo. The spermatological parameters and semen production period (SPP) were higher in cattle. The age at first semen donation and breeding period could be reduced by introducing the bulls to training at an early age. The results revealed an increasing trend in individual motility (IM) while semen volume, AFSC, AFSF, AD, FSPP, SPP, ALSC and ALSF showed a decreasing, however, not a definite trend, over the periods. The semen donation traits like, AFSF, of the cattle and buffalo bulls could be predicted from the AFSC, using prediction equation derived in the present study.

Identification of the MicroRNA Repertoire in TLR-Ligand Challenged Bubaline PBMCs as a Model of Bacterial and Viral Infection

In the present study, we used high-throughput sequencing, miRNA-seq, to discover and explore the expression profiles of known and novel miRNAs in TLR ligand-stimulated vis-à-vis non-stimulated (i.e. Control) peripheral blood mononuclear cells (PBMCs) isolated from blood of healthy Murrah buffaloes. Six small RNA (sRNA) libraries were multiplexed in Ion Torrent PI chip and sequenced on Ion Proton System. The reads obtained were aligned to the Bos taurus genome (UMD3.1 assembly), which is phylogenetically closest species to buffalo (Bubalus bubalis). A total of 160 bovine miRNAs were biocomputationally identified in buffalo PBMCs and 130 putatively novel miRNAs (not enlisted in the bovine mirBase) were identified. All of these 290 miRNAs identified across the six treatment and control samples represent the repertoire of novel miRNAs for the buffalo species. The expression profiles of these miRNAs across the samples have been represented by sample dendrogram and heatmap plots. The uniquely expressed miRNAs in each treatment and control groups were identified. A few miRNAs were expressed at very high levels while the majority of them were moderately expressed. The miRNAs bta-miR-103 and -191 were found to be highly abundant and expressed in all the samples. Other abundantly expressed miRNAs include bta-miR-19b, -29b, -15a, -19a, -30d, -30b-5p and members of let family (let 7a-5p, let 7g & let 7f) in LPS and CpG treated PBMCS and bta-miR-191, -103 & -19b in Poly I:C stimulated PBMCs. Only one novel miRNA (bta-miR-11039) out of 130 identified putatively novel miRNAs, was expressed in all the six samples and differentially expressed (>2- fold) miRNAs were identified. Six of the differentially expressed miRNAs across the groups (bta-miR-421, bta-let-7i, bta-miR-138, bta-miR-21-5p, bta-miR-222 and bta-miR-27b) were subsequently confirmed by TaqMan quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, the target genes of differentially expressed miRNAs were enriched for the roles in innate immunity and TLR signaling pathways. This maiden study on profiling and cataloguing of bubaline miRNAs expressed in TLR-ligand stimulated PBMCs will provide an important reference point for future studies on regulatory roles of miRNAs in immune system of buffaloes.

Biocomputational Characterization and Evolutionary Analysis of Bubaline Dicer1 Enzyme

Dicer, an ribonuclease type III type endonuclease, is the key enzyme involved in biogenesis of microRNAs (miRNAs) and small interfering RNAs (siRNAs), and thus plays a critical role in RNA interference through post transcriptional regulation of gene expression. This enzyme has not been well studied in the Indian water buffalo, an important species known for disease resistance and high milk production. In this study, the primary coding sequence (5,778 bp) of bubaline dicer (GenBank: AB969677.1) was determined and the bubaline Dicer1 biocomputationally characterized to determine the phylogenetic signature among higher eukaryotes. The evolutionary tree revealed that all the transcript variants of Dicer1 belonging to a specific species were within the same node and the sequences belonging to primates, rodents and lagomorphs, avians and reptiles formed independent clusters. The bubaline dicer1 is closely related to that of cattle and other ruminants and significantly divergent from dicer of lower species such as tapeworm, sea urchin and fruit fly. Evolutionary divergence analysis conducted using MEGA6 software indicated that dicer has undergone purifying selection over the time. Seventeen divergent sequences, representing each of the families/taxa were selected to study the specific regions of positive vis-à-vis negative selection using different models like single likelihood ancestor counting, fixed effects likelihood, and random effects likelihood of Datamonkey server. Comparative analysis of the domain structure revealed that Dicer1 is conserved across mammalian species while variation both in terms of length of Dicer enzyme and presence or absence of domain is evident in the lower organisms.

Biocomputational identification and validation of novel microRNAs predicted from bubaline whole genome shotgun sequences

MicroRNAs (miRNAs) are small (19-25 base long), non-coding RNAs that regulate post-transcriptional gene expression by cleaving targeted mRNAs in several eukaryotes. The miRNAs play vital roles in multiple biological and metabolic processes, including developmental timing, signal transduction, cell maintenance and differentiation, diseases and cancers. Experimental identification of microRNAs is expensive and lab-intensive. Alternatively, computational approaches for predicting putative miRNAs from genomic or exomic sequences rely on features of miRNAs viz. secondary structures, sequence conservation, minimum free energy index (MFEI) etc. To date, not a single miRNA has been identified in bubaline (Bubalus bubalis), which is an economically important livestock. The present study aims at predicting the putative miRNAs of buffalo using comparative computational approach from buffalo whole genome shotgun sequencing data (INSDC: AWWX00000000.1). The sequences were blasted against the known mammalian miRNA. The obtained miRNAs were then passed through a series of filtration criteria to obtain the set of predicted (putative and novel) bubaline miRNA. Eight miRNAs were selected based on lowest E-value and validated by real time PCR (SYBR green chemistry) using RNU6 as endogenous control. The results from different trails of real time PCR shows that out of selected 8 miRNAs, only 2 (hsa-miR-1277-5p; bta-miR-2285b) are not expressed in bubaline PBMCs. The potential target genes based on their sequence complementarities were then predicted using miRanda. This work is the first report on prediction of bubaline miRNA from whole genome sequencing data followed by experimental validation. The finding could pave the way to future studies in economically important traits in buffalo.

Improvement in cryosurvival of buffalo bull (Bubalus bubalis) sperm by altering freezing rate within critical temperature range

Objective: To optimize the cryopreservation of buffalo bull semen by altering freezing rates within critical temperature range (4 °C to -60 °C). Methods: A total of 20 ejaculates each from 5 Murrah buffalo bulls were cryopreserved using programmable biofreezer in 2 phases. In the 1st phase, 9 freezing rates were applied at -2, -5, -10, -20, -30, -40, -50, -60 or -4 °C/min (control) from 4 °C to -15 °C ; at -40 °C/min from -15 °C to -60 °C. In the 2nd phase, a fixed freezing rate was applied at -30 °C /min from 4 °C to -15 °C. Six freezing rates were applied at -10, -20, -30, -40 (control), -50 or -60 °C/min from -15 °C to -60 °C. The freezing from -60 °C to -140 °C were fixed at -50 °C/min in both the phases. Post thaw semen quality was assessed in terms of motility, viability, membrane integrity (hypo-osmotic swelling test), sperm abnormalities, and active mitochondria. Data were arc sine transformed and analyzed through one-way analysis of variance using SPSS software. Results: In the 1st phase, percent individual motility, progressive motility and viability were similar among various protocols. Percent hypo-osmotic swelling reactive sperm was higher with freezing at -30 °C/min. In the 2nd phase, percent individual motility, viability and hypo-osmotic swelling reactive sperm was higher with freezing at -50 °C /min. Sperm head abnormalities were lower at -30 °C /min in the 1st phase, but were similar among the protocols of the 2nd phase. Percent active mitochondria were higher at -30 °C /min in the 1st phase and at -50 °C/min in the 2nd phase. Conclusions: The optimum post thaw semen quality of buffalo bull could be obtained by applying freezing rate at -30 °C/min (4 °C to -15 °C) and at -50 °C/min (-15 °C to -140 °C ), followed by plunging of straws in into liquid nitrogen for storage.