Project description:The domestic buffalo (Bubalus bubalis) has presented an important role in the livestock industry, contributing to milk and meat production worldwide, especially in developing countries. However, little is known about its reproductive particularities. Studies regarding protein composition of buffalo SP are still limited and a complete mapping of buffalo SP proteins is still lacking in the literature. Hence, a comprehensive study of SP proteome is of great importance to better understand the mechanisms involved in male reproduction and to optimize the reproductive biotechnologies of farm animal species. Therefore, the aim of this study is to describe for the first time the Bubalus bubalis seminal plasma proteome using a label free shotgun HDMS approach. This type of analysis is interesting since it yields a high number of detected proteins, generating a dataset that is useful for further characterizing the buffalo SP.
Project description:The study reports a differential proteomic analysis of the Mediterranean buffalo milk to evaluate the changes induced by Staphylococcus spp. during a subclinical intramammary infection (IMI). A number of 12 quarter milk samples, 6 of which with somatic cell count (SCC) < 50,000 cells/mL and culture-negative, and the other 6 with SCC ˃ 3,000,000 cells/mL and culture-positive to Staphylococcus aureus (SAU, n=3), SAU, or non-aureus staphylococci (NAS, n=3) was selected. Samples were analyzed using a shotgun proteomics approach, based on filter-aided sample preparation (FASP) followed by LC-MS/MS and label-free analysis. Here, the largest buffalo milk protein dataset described so far was reported. Moreover, the results demonstrated that staphylococcal IMI mostly affected proteins involved in structural functions and in innate immune defense, with changes in their abundance that were generally more intense in SAU than in NAS samples. Further, an increase in the abundance of different cathelicidins was observed, as already reported for other animals with mastitis disease (1,2). (1) Addis MF, Pisanu S, Marogna G, Cubeddu T, Pagnozzi D, Cacciotto C, et al. Production and release of antimicrobial and immune defense proteins by mammary epithelial cells following Streptococcus uberis infection of sheep. Infect Immun. 2013;81: 3182–3197. (2) Addis MF, Tedde V, Dore S, Pisanu S, Puggioni GMG, Roggio AM, et al. Evaluation of milk cathelicidin for detection of dairy sheep mastitis. J Dairy Sci. Elsevier; 2016;99: 6446–6456. In conclusion, our results provide the first in depth characterization of buffalo milk proteins, describe the changes induced by SAU and NAS subclinical intramammary infection and suggest indications to reveal subclinical staphylococcal mastitis in buffalo by the milk proteome investigation.
Project description:Infectious mastitis by non-aureus staphylococci (NAS) is a significant issue in dairy buffalo farming. In a herd with subclinical NAS mastitis, we identified Staphylococcus microti as the predominant species by MALDI-TOF mass spectrometry. To assess milk protein integrity and investigate potential disease markers, we characterized 12 NAS-positive and 12 healthy quarter milk samples by shotgun peptidomics combining peptide enrichment and high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS). We observed significant changes in the milk peptidome. Out of 789 total peptides identified in each group, 49 and 44 were unique or increased in NAS-positive and healthy milk, respectively. In terms of sequence, these belonged mainly to caseins in NAS-positive milk, followed by milk fat globule membrane proteins (MFGMP) and by the immune defense/antimicrobial proteins osteopontin, lactoperoxidase, and serum amyloid A. In healthy milk, the differential peptides belonged mainly to MFGMP, followed by caseins. In terms of abundance, peptides from MFGMP and immune defense protein were higher in NAS-positive milk, while peptides from caseins were higher in healthy milk. These findings highlight the impact of NAS on buffalo milk quality and mammary gland health, even when clinical signs are not evident, and underscore the need for clarifying the epidemiology and relevance of the different NAS species in this dairy ruminant.
Project description:Duplicated sequences are the important source of gene innovation and structural variation within mammalian genomes. Using a read depth approach based on next-generation sequencing, we performed a genome-wide analysis of segmental duplications (SDs) and associated copy number variants (CNVs) in water buffalo (Bubalus bubalis). Aligning to the UMD3.1 cattle genome, we estimated 44.6 Mb (~1.73% of cattle genome) segmental duplications in the autosomes and X chromosome using the sequencing reads of Olimpia (the sequenced water buffalo). 70.3% (70/101) duplications were experimentally validated using the fluorescent in situ hybridization. We also detected a total of 1344 CNV regions across 14 additional water buffalos as well as Olimpia, amounting to 59.8Mb of variable sequence or 2.2% of the cattle genome. The CNV regions overlap 1245 genes and are significantly enriched for specific biological functions such as immune response, oxygen transport, sensory system and signalling transduction. Additionally, we performed array Comparative Genomic Hybridization (aCGH) experiments using the 14 water buffalos as test samples and Olimpia as the reference. Using a linear regression model, significant and high Pearson correlations (r = 0.781) were observed between the digital aCGH values and aCGH probe log2 ratios. We further designed Quantitative PCR assays to confirm CNV regions within or near annotated genes and found 74.2% agreement with our CNV predictions.
Project description:Gonadotropin surge acts on the preovulatory follicle of the ovary to induce luteinization of follicular cells, oocyte meiotic maturation, cumulus expansion and follicular rupture leading to ovulation. These processes are brought about by spatial and temporal changes in transcriptional regulation of genes in the follicular cells in response to the gonadotropin surge. Analysis of gene expression changes in the periovulatory follicular cells will help in delineating the signal transduction pathways involved in the above mentioned processes. In monoovulatory species like bovines, the time interval of 24-28 hours between gonadotropin surge and ovulation provides distinct advantage for studying the temporal changes in the gene expression pattern. Thus, in the present study, we attempt to identify the temporal changes in the global gene expression profile in the periovulatory follicle of buffalo cows in response to gonadotropin surge and the results suggest the involvement of Insulin-like Growth Factor 1 and cytokine signaling pathways in the periovulatory events. Experiment Overall Design: To study the periovulatory gene expression changes in buffalo cows, an induced-ovulation model system involving sequential treatment with PGF2alpha and GnRH was standardized. The follicular wave containing at least one large follicle of ~7mm size was determined by ultrasonography on day 7 of the estrous cycle before administering exogenous PGF2alpha to induce luteolysis and follicular growth. Exogenous GnRH (100µg i.m) was administered 36h post PGF2alpha to induce LH surge. The time course of increase in LH levels post GnRH injection was monitored. Since peak LH levels are attained 2 h post GnRH administration, the time intervals of 3 h post GnRH (corresponding to1 h post LH surge) and 24 h post GnRH (corresponding to 22 h post LH surge) were chosen to identify the gene expression profile associated with immediate early and delayed changes in periovulatory follicle respectively. Thus ovaries were collected before, 1 h and 22 h post LH surge and follicle wall and granulosa cells were isolated from the ovaries and snap frozen for the purpose of RNA isolation.