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Beneficial effects of n-3 polyunsaturated fatty acid (PUFA) supplementation on dairy cow reproduction have been previously reported. The objectives of the present study were to assess whether n-3 PUFA supplementation would affect in vitro embryo production (IVP) after ovarian stimulation. Holstein cows received a diet with 1% dry matter supplementation of either n-3 PUFA (n = 18, microencapsulated fish oil) or a control, n-6 PUFA (n = 19, microencapsulated soy oil). Both plasma and follicular fluid FA composition showed integration of total PUFA through the diet. All cows underwent an IVP protocol consisting of ovarian stimulation, ultrasound-guided transvaginal oocyte retrieval (ovum pick-up, OPU, five per cow) followed by in vitro maturation, fertilisation and 7 days of embryo development. A tendency toward an increase in the blastocyst rate (diet effect, P = 0.0865) was observed in n-3 cows, with 49.6 ± 5.5% vs 42.3 ± 5.5% in control n-6 cows. A significant increase (diet effect, P = 0.0217) in the good-quality blastocyst rate (freezable blastocysts) was reported in n-3 cows (42.2 ± 7.7%) compared to control n-6 cows (32.7 ± 7.7%). A significant difference in lipid composition was shown in the oocytes recovered by OPU from n-3 and n-6 treated cows, by intact single-oocyte MALDI-TOF mass spectrometry. The 42 differentially abundant identified lipids were mainly involved in cell membrane structure. In conclusion, n-3 PUFA supplementation enhanced oocyte quality and modified their lipid composition. Further studies are necessary to investigate the potential link of these lipid modifications with enhanced oocyte quality.

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The endogenous peptides and small proteins present in chicken sperm were identified in the context of the characterization of a fertility-diagnostic method based on the use of ICM-MS (Intact Cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry). The interpretation and description of these data can be found in a research article, "Intact cell MALDI-TOF MS on sperm: a molecular test for male fertility diagnosis" (Soler et al., 2016) [1], and raw data derived from this analysis have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002768. Here, we describe the inventory of all the molecular species identified, along with their biochemical features and functional analysis. This peptide/protein catalogue can be further employed as reference for other studies and reveal that the use of proteomics allows for a global evaluation of sperm cells functions.

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Photoperiod is the main physical synchronizer of seasonal functions and a key factor in the modulation of molecule access to cerebrospinal fluid (CSF) in animals. Previous work has shown that photoperiod affects the transfer rate of steroids and protein hormones from blood to CSF and modulates choroid plexus tight junction protein content. We hypothesized that the CSF proteome would also be modified by photoperiod. We tested this hypothesis by comparing CSF obtained from the third ventricle of mature, ovariectomized, estradiol-replaced ewes exposed to long day length (LD) or short day length (SD). Variations in CSF protein expression between SD- or LD-treated ewes were studied in pools of CSF collected for 48 h. Proteins were precipitated, concentrated, and included in a polyacrylamide gel without protein fractionation. After in-gel tryptic digestion of total protein samples, we analyzed the resulting peptides by nanoliquid chromatography coupled with high-resolution tandem mass spectrometry (GeLC-MS/MS). Quantitative analysis was performed using 2 methods based on spectral counting and extracted ion chromatograms. Among 103 identified proteins, 41 were differentially expressed between LD and SD ewes (with P < 0.05 and at least a 1.5-fold difference). Of the 41 differentially expressed proteins, 22 were identified by both methods and 19 using extracted ion chromatograms only. Eighteen proteins were more abundant in LD ewes and 23 were more abundant in SD ewes. These proteins are involved in numerous functions including hormone transport, immune system activity, metabolism, and angiogenesis. To confirm proteomic results, 2 proteins, pigment epithelium-derived factor (PEDF) and gelsolin, for each individual sample of CSF collected under SD or LD were analyzed with Western blots. These results suggest an important photoperiod-dependent change in CSF proteome composition. Nevertheless, additional studies are required to assess the role of each protein in seasonal functions.

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The Brucella melitensis sucB gene encoding the dihydrolipoamide succinyltransferase (E2o) enzyme (previously identified as an immunogenic protein in infected sheep) was cloned and sequenced. The amino acid sequence predicted from the cloned gene revealed 88.8 and 51.2% identity to the dihydrolipoamide succinyltransferase SucB protein from Brucella abortus and Escherichia coli, respectively. Sera from naturally infected sheep showed antibody reactivity against the recombinant SucB protein.

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Brucella melitensis is a facultative intracellular pathogen which is able to survive and replicate within phagocytic cells. Therefore, it has to adapt to a range of different hostile environments. In order to understand the mechanisms of intracellular survival employed by virulent B. melitensis 16M, an initial approach consisting of analysis of the differences in patterns of protein synthesis in response to heat, oxidative, and acid pH stresses by two-dimensional (2-D) polyacrylamide gel electrophoresis was used. Depending on the stress, this involved about 6.4 to 12% of the 676 protein spots detected in 2-D gel electrophoresis. On the basis of N-terminal sequence analysis and database searching, 19 proteins whose level of synthesis was up- or down-regulated by stress conditions were identified. Some of them were previously reported for Brucella, such as BvrR, DnaK, GroEL, and Cu-Zn superoxide dismutase (SOD). Eight other proteins closely matched proteins found in other bacteria: AapJ, alpha-ETF, ClpP, Fe and/or Mn SOD, malate dehydrogenase, IalB, 30S ribosomal protein S1, and pyruvate dehydrogenase E1 component beta subunit. Results indicated that B. melitensis could bring specific regulatory mechanisms into play in response to stress conditions. For example, the ribosome releasing factor in B. melitensis appeared to be a heat shock protein, whereas the ClpP protein, described as a heat shock protein for Escherichia coli, was strongly down-regulated in B. melitensis in response to heat stress. Some of the identified proteins and their potential specific regulation could be required for the adaptation of B. melitensis to environmental stresses encountered in phagocytic cells and possibly for bacterial virulence.

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In a previous report, proteins from Brucella melitensis were characterized by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and N-terminal microsequencing. In the present report, we have extended this study to the second etiologic agent in ovine brucellosis, B. ovis, responsible for ram epididymitis and infertility. The combination of 2-D gel electrophoresis and protein microsequencing facilitated the location and identification of the major proteins of B. ovis on the 2-D pattern. These proteins comprised cytoplasmic, periplasmic, and some membrane proteins except the major outer membrane proteins. By comparing 2-D gel profiles of B. ovis with that of B. melitensis described previously, a few proteins with different expression levels were readily identified. Serum from a ram naturally infected with B. ovis was used in immunoblotting studies to identify immunogenic proteins recognized during the course of infection. This serum showed antibody reactivity against approximately 82 protein spots. Twenty-one of these proteins were identified either by use of monoclonal antibodies or by N-terminal microsequencing. Several proteins previously described in earlier Brucella works were identified: the 89 kDa outer membrane protein, DnaK, GroEL, BP26, and Cu-Zn superoxide dismutase. Eight proteins had amino acid sequences homologous to those of various proteins from other bacteria found in protein databases; NikA, dihydrolipoamide succinyltransferase, a hypothetical 31 kDa protein, malate dehydrogenase, succinyl-CoA synthetase alpha subunit, an amino acid ABC type transporter, Leu/Ile/Val-binding protein precursor, and ClpP. The remaining eight proteins had N-terminal sequences lacking similarity to existing databases entries. Thus, the 2-D PAGE analysis provided a convenient first approach in the characterization of immunogenic proteins.

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Two-dimensional (2-D) gel electrophoresis was used to map Brucella melitensis proteins. The 2-D proteins map of B. melitensis B115 revealed 595 silver-stained protein spots separated by both isoelectric point and molecular mass. Twenty-five proteins were identified either by immunoblotting using monoclonal antibodies (MAbs) or by N-terminal microsequencing. The protein spots identified by MAbs were the 89 kDa outer membrane protein, DnaK, bacterioferritin, CP24, and BP26. Some spots were identified by N-terminal microsequencing as proteins whose sequences had been reported previously from Brucella, such as three heat-shock proteins, namely DnaK, GroEL and GroES; bacterioferritin; Cu-Zn superoxide dismutase; and the 50S ribosomal protein L7/L12. Other proteins had amino acid sequences homologous with those of various proteins from other bacteria found in protein databases: ClpP; the 10K-S protein; the ORFU phosphoprotein; succinyl-CoA synthetase alpha sub-unit; an inorganic pyrophosphatase; the Fe and/or Mn superoxide dismutase; the nucleoside diphosphate kinase, an amino acid ABC type transporter, and an electron transfer flavoprotein small subunit. Seven proteins were identified with N-terminal sequences not yet reported in databases. The 2-D map established in this study will be the basis for comparative studies of protein expression in Brucella.

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