Cryptosporidium parvum infection alters the intestinal mucosa transcriptome in neonatal calves: implications for immune function

One of the leading causes of infectious diarrhea in newborn calves is the apicomplexan protozoan Cryptosporidium parvum (C. parvum). However, little is known about its immunopathogenesis. Using next generation sequencing, this study investigated the immune transcriptional response to C. parvum infection in neonatal calves. Neonatal male Holstein-Friesian calves were either orally infected (N = 5) or not (CTRL group, N = 5) with C. parvum oocysts (gp60 subtype IIaA15G2R1) at day 1 of life and slaughtered on day 7 after infection. Total RNA was extracted from the jejunal mucosa for short read. Differentially expressed genes (DEGs) between infected and CTRL groups were assessed using DESeq2 at a false discovery rate < 0.05. Infection did not affect plasma immunohematological parameters, including neutrophil, lymphocyte, monocyte, leucocyte, thrombocyte, and erythrocyte counts as well as hematocrit and hemoglobin concentration on day 7 post infection. The immune-related DEGs were selected according to the UniProt immune system process database and were used for gene ontology (GO) and pathway enrichment analysis using Cytoscape (v3.9.1). Based on GO analysis, DEGs annotated to mucosal immunity, recognizing and presenting antigens, chemotaxis of neutrophils, eosinophils, natural killer cells, B and T cells mediated by signaling pathways including toll like receptors, interleukins, tumor necrosis factor, T cell receptor, and NF-KB were upregulated, while markers of macrophages chemotaxis and cytosolic pattern recognition were downregulated. This study provides a holistic snapshot of immune-related pathways induced by C. parvum in calves, including novel and detailed feedback and feedforward regulatory mechanisms establishing the crosstalk between innate and adaptive immune response in neonate calves, which could be utilized further to develop new therapeutic strategies

Longitudinal liver proteome profiling in dairy cows during the transition from gestation to lactation: Investigating metabolic adaptations and their interactions with fatty acids supplementation via repeated measurements ANOVA-simultaneous component analysis

International audienceRepeated measurements analysis of variance - simultaneous component analysis (ASCA) has been developed to handle complex longitudinal omics datasets and combine novel information with existing data. Herein, we aimed at applying ASCA to 64 liver proteomes collected at 4-time points (day -21, +1, +28, and + 63 relative to parturition) from 16 Holstein cows treated from 9 wk. antepartum to 9 wk. postpartum (PP) with coconut oil (CTRL) or a mixture of essential fatty acids (EFA) and conjugated linoleic acid (CLA) (EFA + CLA). The ASCA modeled 116, 43, and 97 differentially abundant proteins (DAP) during the transition to lactation, between CTRL and EFA + CLA, and their interaction, respectively. Time-dependent DAP were annotated to pathways related to the metabolism of carbohydrates, FA, and amino acid in the PP period. The DAP between FA and the interaction effect were annotated to the metabolism of xenobiotics by cytochrome P450, drug metabolism - cytochrome P450, retinol metabolism, and steroid hormone biosynthesis. Collectively, ASCA provided novel information on molecular markers of metabolic adaptations and their interactions with EFA + CLA supplementation. Bioinformatics analysis suggested that supplemental EFA + CLA amplified hepatic FA oxidation; cytochrome P450 was enriched to maintain metabolic homeostasis by oxidation/detoxification of endogenous compounds and xenobiotics.Significance: This report is among the first ones applying repeated measurement analysis of variance-simultaneous component analysis (ASCA) to deal with longitudinal proteomics results. ASCA separately identified differentially abundant proteins (DAP) in 'transition time', 'between fatty acid treatments', and 'their interaction'. We first identified the molecular signature of hepatic metabolic adaptations during postpartum negative energy balance; the enriched pathways were well-known pathways related to mobilizing fatty acids (FA) and amino acids to support continuous energy production through fatty acid oxidation, TCA cycle, and gluconeogenesis. Some of the DAP were not previously reported in transition dairy cows. Secondly, we provide novel information on the mechanisms by which supplemented essential FA and conjugated linoleic acids interact with hepatic metabolism. In this regard, FA amplified hepatic detoxifying and oxidation capacity through ligand activation of nuclear receptors. Finally, we briefly compared the strengths and weaknesses of the ASCA model with PLS-DA and outlined why these methods are complementary

Plasma proteomics reveals crosstalk between lipid metabolism and immunity in dairy cows receiving essential fatty acids and conjugated linoleic acid

International audienceAbstract Essential fatty acids (EFA) and conjugated linoleic acids (CLA) are unsaturated fatty acids with immune-modulatory effects, yet their synergistic effect is poorly understood in dairy cows. This study aimed at identifying differentially abundant proteins (DAP) and their associated pathways in dairy cows supplied with a combination of EFA and CLA during the transition from antepartum (AP) to early postpartum (PP). Sixteen Holstein cows were abomasally infused with coconut oil as a control (CTRL) or a mixture of EFA (linseed + safflower oil) and CLA (Lutalin, BASF) (EFA + CLA) from − 63 to + 63 days relative to parturition. Label-free quantitative proteomics was performed on plasma samples collected at days − 21, + 1, + 28, and + 63. During the transition time, DAP, consisting of a cluster of apolipoproteins (APO), including APOE, APOH, and APOB, along with a cluster of immune-related proteins, were related to complement and coagulation cascades, inflammatory response, and cholesterol metabolism. In response to EFA + CLA, specific APO comprising APOC3, APOA1, APOA4, and APOC4 were increased in a time-dependent manner; they were linked to triglyceride-enriched lipoprotein metabolisms and immune function. Altogether, these results provide new insights into metabolic and immune adaptation and crosstalk between them in transition dairy cows divergent in EFA + CLA status

Effect of Linolenic acid during in vitro maturation of ovine oocytes: embryonic developmental potential and mRNA abundances of genes involved in apoptosis

To study the effect of alpha-linolenic acid (ALA) on meiotic maturation, mRNA abundance of apoptosis-related (Bax and Bcl-2) molecules, and blastocyst formation in ovine oocytes.Purpose To study the effect of &alpha;-linolenic acid (ALA) on meiotic maturation, mRNAabundance of apoptosis-related (Bax and Bcl-2) molecules, and blastocyst formation inovine oocytes.Methods A preliminary experiment was conducted to analyze the concentration of ALAin &quot;small&quot; (&le;2 mm) and &quot;large&quot; (&ge;6 mm) follicles using gas chromatography/massspectrometry analysis. The concentration of ALA in small and large follicles wasdetermined to be in a range of 75.4 to 125.7 &mu;M, respectively. In vitro maturation (IVM)of oocyte was then performed in presence of 0 (control), 10 (ALA-10), 50 (ALA-50),100 (ALA-100), and 200 (ALA-200) &mu;M of ALA. Meiotic maturation and mRNAabundance of Bax, and Bcl-2 genes was evaluated after 24 h of IVM. The embryoniccleavage and blastocyst formation following parthenogenetic activation were alsodetermined for each group.Results The highest concentration of ALA (ALA-200) decreased the oocyte maturationrate compared with the control group. Analysis of apoptosis-related genes in oocytesafter IVM revealed lesser transcript abundances for Bax gene, and higher transcriptabundances for Bcl-2 gene in ALA-treated oocytes as compared with the controloocytes. In term of cleavage rate (considered as 2-cell progression), we did notobserve any differences among the groups. However, ALA-100 group promoted moreblastocyst formation as compared with the control group.Conclusion Our results suggested that ALA treatment during IVM had a beneficial effect on developmental competence of ovine oocytes by increasing the blastocystformation and this might be due to the altered abundance of apoptosis-regulatorygenes.</p

Liver proteome profiling in dairy cows during the transition from gestation to lactation: Effects of supplementation with essential fatty acids and conjugated linoleic acids as explored by PLS-DA

International audienceThis study aimed at investigating the synergistic effects of essential fatty acids (EFA) and conjugated linoleic acids (CLA) on the liver proteome profile of dairy cows during the transition to lactation. 16 Holstein cows were infused from 9 wk. antepartum to 9 wk. postpartum into the abomasum with either coconut oil (CTRL) or a mixture of EFA (linseed + safflower oil) and CLA (EFA + CLA). Label-free quantitative proteomics was performed in liver tissue biopsied at days -21, +1, +28, and + 63 relative to calving. Differentially abundant proteins (DAP) between treatment groups were identified at the intersection between a multivariate and a univariate analysis. In total, 1680 proteins were identified at each time point, of which between groups DAP were assigned to the metabolism of xenobiotics by cytochrome P450, drug metabolism - cytochrome P450, steroid hormone biosynthesis, glycolysis/gluconeogenesis, and glutathione metabolism. Cytochrome P450, as a central hub, enriched with specific CYP enzymes comprising: CYP51A1 (d - 21), CYP1A1 & CYP4F2 (d + 28), and CYP4V2 (d + 63). Collectively, supplementation of EFA + CLA in transition cows impacted hepatic lipid metabolism and enriched several common biological pathways at all time points that were mainly related to ω-oxidation of fatty acids through the Cytochrome p450 pathway. SIGNIFICANCE: In three aspects this manuscript is notable. First, this is among the first longitudinal proteomics studies in nutrition of dairy cows. The selected time points are critical periods around parturition with profound endocrine and metabolic adaptations. Second, our findings provided novel information on key drivers of biologically relevant pathways suggested according to previously reported performance, zootechnical, and metabolism data (already published elsewhere). Third, our results revealed the role of cytochrome P450 that is hardly investigated, and of ω-oxidation pathways in the metabolism of fatty acids with the involvement of specific enzymes

Intracellular glutathione content, developmental competence and expression of apoptosis-related genes associated with G6PDH-activity in goat oocyte

To associate glucose-6-phosphate dehydrogenase (G6PDH) activity in goat oocytes with intracellular glutathione (GSH) content, meiotic competence, developmental potential, and relative abundance of Bax and Bcl-2 genes transcripts.Purpose To associate the glucose-6-phosphate dehydrogenase (G6PDH) activity inimmature goat oocyte with intracellular glutathione (GSH) content, developmentalcompetence after parthenogenetic activation and relative abundance of Bax and Bcl-2genes transcripts using .Methods Goat oocytes were exposed to brilliant cresyl blue (BCB) staining andcategorized into BCB+ (blue cytoplasm) and BCB&minus; (colorless cytoplasm) groups. Agroup of oocytes were not exposed to BCB stain and was considered as a control.After maturation in vitro, a group of oocytes were used for determination of nuclearstatus and intracellular GSH content and another group were subjected toparthenogenetic activation followed by in vitro embryo culture.Results We found that BCB+ oocytes yielded a significantly higher maturation rate thanthe BCB&minus; and control oocytes. Moreover, BCB+ oocytes showed increased inintracellular level of GSH than BCB&minus; and control oocytes. Furthermore, BCB+ oocytesproduced more blastocysts than BCB&minus; and control oocytes. The expression of antiapoptoticgene Bcl-2 and pro-apoptotic gene Bax in mature oocyte, their surroundingcumulus cells and blastocyst was affected by BCB staining test.Conclusions These results strongly suggest that BCB+ oocytes have a higher meioticand cytoplasmic maturity (as determined by intracellular GSH content), and that BCBstaining can be used to select developmentally competent oocytes for in vitro embryoproduction.</p

Association of glucose-6-phosphate dehydrogenase activity with oocyte cytoplasmic lipid content, developmental competence, and expression of candidate genes in a sheep model

To evaluate associations of glucose-6-phosphate dehydrogenase (G6PDH) activity in sheep oocytes with cytoplasmic lipid content, maturational competence, developmental competence to the blastocyst stage, and gene expression of certain molecular markers.Purpose To evaluate associations of glucose-6-phosphate dehydrogenase (G6PDH)activity in sheep oocytes with cytoplasmic lipid content, maturational competence,developmental competence to the blastocyst stage, and gene expression of certainmolecular markers.Methods Before brilliant cresyl blue (BCB) staining test, oocytes were classified ashigh, middle, and low cytoplasmic lipid content (HCLC, MCLC, and LCLC) and after thetest as having low or high G6PDH-activity (BCB+ and BCB-, respectively). Aftermaturation in vitro, a group of oocytes were subjected to IVF followed by in vitroembryo culture and another group was used for evaluation of expression of candidategenes.Results The cleavage and blastosyst rates were lowest (P &lt; 0.05) in LCLC group,intermediate (P &lt; 0.05) in MCLC group and highest (P &lt; 0.05) in HCLC group. More(P &lt; 0.05) oocytes in HCLC group were BCB+, and higher (P &lt; 0.05) maturation,cleavage, and blastocyst rates were seen for BCB+ oocytes than the BCB- oocytes.Our gene expression data indicated that mRNA transcript abundance of ITGB2, pZP3,BMP15, and GDF9 genes were similar between BCB oocytes groups. However, theexpression of ATP1A1 was higher (P &lt; 0.05) for BCB+ oocytes compared to BCBoocytes.In addition, BAX transcript abundance was similar (P &gt; 0.05) among BCB+,BCB-, and control groups, before and after maturation in vitro.Conclusion Activity of G6PDH in sheep oocytes is highly associated with lipid content,and compared with the morphological parameters, might be a more precise andobjective predictor for subsequent developmental competence in vitro.</p