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.

Review: Effect of essential fatty acids and conjugated linoleic acid on the adaptive physiology of dairy cows during the transition period.

Cows fed total mixed rations (silage-based) may not receive as much essential fatty acids (EFAs) and conjugated linoleic acids (CLAs) as cows fed pasture-based rations (fresh grass) containing rich sources of polyunsaturated fatty acids. CLA-induced milk fat depression allows dairy cows to conserve more metabolisable energy, thereby shortening the state of negative energy balance and reducing excessive fat mobilisation at early lactation. EFAs, particularly α-linolenic acid, exert anti-inflammatory and antioxidative properties, thereby modulating immune functions. Thus, combined EFA and CLA supplementation seems to be an effective nutritional strategy to relieve energy metabolism and to improve immune response, which are often compromised during the transition from late pregnancy to lactation in high-yielding dairy cows. There has been extensive research on this idea over the last two decades, and despite promising results, several interfering factors have led to varying findings, making it difficult to conclude whether and under what conditions EFA and CLA supplementations are beneficial for dairy cows during the transition period. This article reviews the latest studies on the effects of EFA and CLA supplementation, alone or in combination, on dairy cow metabolism and health during various stages around parturition. Our review article summarises and provides novel insights into the mechanisms by which EFA and/or CLA influence markers of metabolism, energy homeostasis and partitioning, immunity, and inflammation revealed by a deep molecular phenotyping.

Investigating circulating miRNA in transition dairy cows: What miRNAomics tells about metabolic adaptation.

In the current study, we investigated dairy cows' circulating microRNA (miRNA) expression signature during several key time points around calving, to get insights into different aspects of metabolic adaptation. In a trial with 32 dairy cows, plasma samples were collected on days -21, 1, 28, and 63 relative to calving. Individually extracted total RNA was subjected to RNA sequencing using NovaSeq 6,000 (Illumina, CA) on the respective platform of IGA Technology Services, Udine, Italy. MiRDeep2 was used to identify known and novel miRNA according to the miRbase collection. Differentially expressed miRNA (DEM) were assessed at a threshold of fold-change > 1.5 and false discovery rate < 0.05 using the edgeR package. The MiRWalk database was used to predict DEM targets and their associated KEGG pathways. Among a total of 1,692 identified miRNA, 445 known miRNA were included for statistical analysis, of which 84, 59, and 61 DEM were found between days -21 to 1, 1 to 28, and 28 to 63, respectively. These miRNA were annotated to KEGG pathways targeting the insulin, MAPK, Ras, Wnt, Hippo, sphingolipid, T cell receptor, and mTOR signaling pathways. MiRNA-mRNA network analysis identified miRNA as master regulators of the biological process including miR-138, miR-149-5p, miR-2466-3p, miR-214, miR-504, and miR-6523a. This study provided new insights into the miRNA signatures of transition to the lactation period. Calving emerged as a critical time point when miRNA were most affected, while the following period appeared to be recovering from massive parturition changes. The primarily affected pathways were key signaling pathways related to establishing metabolic and immune adaptations.

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

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.

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.

This 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.

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.

Repeated 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.

Maternal supplementation with fish oil modulates inflammation-related MicroRNAs and genes in suckling lambs.

Dietary n-3 long-chain fatty acids (n-3 LCFA) have been shown to modify lipid metabolism and immune function. The objective of this study was to evaluate the effect of periparturient fish oil (FO) supplementation on the inflammation and metabolic health of ewes and their lambs at a molecular level. Prepartum ewes were fed control diet (CON, n = 12) or CON supplemented with 2% DM of calcium soap of FO (n = 12) from 28 days before until 21 days after parturition. The ewes were evaluated for plasma metabolites and milk composition. The experiment was followed by analyzing the relative transcript abundance of circulating microRNAs (miRNAs) in plasma and targeted miRNA/mRNA expression in peripheral blood mononuclear cells (PBMCs) in both ewes and lambs. FO treatment decreased prepartum feed intake (1812 ± 35 vs 1674 ± 33 g/day, P < 0.01), whereas the influence on plasma metabolites was negligible. Dietary FO supplementation decreased milk fat percentage (8.82 ± 0.49 vs 7.03 ± 0.45, P = 0.02) and reduced milk n-6/n-3 (P < 0.05). Also, it altered the expression of plasma-circulating miRNAs in both ewe and lamb (P < 0.05). Furthermore, maternal nutrition of FO downregulated the relative expression of miR-33a and miR-146b and transcript abundance of genes IL-1ß (0.41-fold) and NF-κB (0.25-fold) in lambs' PBMC. In conclusion, results showed that FO supplementation starting antepartum affects milk composition and circulating miRNA in dams and the inflammatory markers in lambs delivered by the supplemented ewes. These may provide a strategy to maintain immune balance during gestation and develop the immune system in lambs.

Oocyte maturation with royal jelly increases embryo development and reduces apoptosis in goats.

Royal jelly (RJ) was supplemented to goat oocyte in vitro maturation (IVM) medium at three different concentrations (2.5, 5, and 10 mg/ml). Maturation rate, embryo cleavage, and blastocyst rate were recorded. Gene expression of apoptosis-related transcripts was investigated in matured oocytes. Percentage of oocytes that reached MII-stage was increased in RJ-treated groups compared to the control group. Glutathione (GSH) content of mature oocytes was enhanced when RJ was added to IVM medium at any supplementation compared with control. Percentage of cleaved embryos and blastocysts was higher in the RJ-treated groups at a concentration of 5 than in the 2.5 mg/ml and control group. Total number of cells per blastocyst was not different in the control and RJ-treated group at 5 mg/ml. However, number of apoptotic cells per blastocyst was higher in the control group than in the RJ-treated group at 5 mg/ml. Expression profile of Bax, and p53 was down-regulated while Bcl-2 was up-regulated in oocytes treated with RJ at 5 and 10 mg/ml compared with the control group. Addition of RJ at concentrations of 5 mg/ml improved embryo production through increasing maturation rate. RJ seems to improve the IVM microenvironment by reducing expression of genes inducing apoptosis, enhancing GSH content, and reducing incidence of apoptosis in blastocysts.

Improved stem cell therapy of spinal cord injury using GDNF-overexpressed bone marrow stem cells in a rat model.

The use of stem cell base therapy as an effective strategy for the treatment of spinal cord injury (SCI) is very promising. Although some strategy has been made to generate neural-like cells using bone marrow mesenchymal stem cells (BMSCs), the differentiation strategies are still inefficiently. For this purpose, we improved the therapeutic outcome with utilize both of N-neurotrophic factor derived Gelial cells (GDNF) gene and differentiation medium that induce the BMSCs into the neural-like cells. The differentiated GDNF overexpressed BMSCs (BMSCs-GDNF) were injected on the third day of post-SCI. BBB score test was performed for four weeks. Two weeks before the end of BBB, biotin dextranamin was injected intracrebrally and at the end of the fourth week, the tissue was stained. BBB scores were significantly different in BMSCs-GDNF injected and control animals. Significant difference in axon counting was observed in BMSCs-GDNF treated animals compared to the control group. According to the results, differentiated BMSCs-GDNF showed better results in comparison to the BMSCs without genetic modification. This study provides a new strategy to investigate the role of simultaneous in stem cell and gene therapy for future neural-like cells transplantation base therapies for SCI.

Linolenic acid improves oocyte developmental competence and decreases apoptosis of in vitro-produced blastocysts in goat.

The effects of α-linolenic acid (ALA) on developmental competence of oocytes in goats were evaluated in this study. Initially, the level of ALA in small and large antral follicles was determined to be in a range of 0.018-0.028 mg/ml (64.6-100.6 µM, respectively). In vitro maturation was performed in the presence of various concentrations (10, 50, 100, or 200 µM) of ALA. Cumulus expansion, meiotic maturation, levels of intracellular glutathione (GSH), embryonic cleavage, blastocyst formation following parthenogenetic activation (PA) and in vitro fertilization (IVF), number of total and apoptotic cells in blastocyst, and expression of Bax, Bcl-2, and p53 genes in blastocyst cells were determined. Compared with the control, no improvement was observed in cumulus expansion in ALA-treated groups. At 50 µM concentration, ALA increased meiotic maturation rate but had no effect on GSH level. When oocytes treated with 50 µM ALA were subsequently used for PA or IVF, a higher rate of blastocyst formation was observed, and these embryos had a higher total cell number and a lower apoptotic cell number. Expression analyses of genes in blastocysts revealed lesser transcript abundances for Bax gene, and higher transcript abundances for Bcl-2 gene in 50 µM ALA group. Expression of p53 gene was also less observed in ALA-treated blastocysts. Our results show that ALA treatment at 50 µM during in vitro maturation (IVM) had a beneficial effect on maturation of goat oocytes and this, in turn, stimulated embryonic development and regulated apoptotic gene expression.

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

PURPOSE: To study the effect of α-linolenic acid (ALA) on meiotic maturation, mRNA abundance of apoptosis-related (Bax and Bcl-2) molecules, and blastocyst formation in ovine oocytes. METHODS: A preliminary experiment was conducted to analyze the concentration of ALA in "small" (≤2 mm) and "large" (≥6 mm) follicles using gas chromatography/mass spectrometry analysis. The concentration of ALA in small and large follicles was determined to be in a range of 75.4 to 125.7 µM, 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) µM of ALA. Meiotic maturation and mRNA abundance of Bax, and Bcl-2 genes was evaluated after 24 h of IVM. The embryonic cleavage and blastocyst formation following parthenogenetic activation were also determined for each group. RESULTS: The highest concentration of ALA (ALA-200) decreased the oocyte maturation rate compared with the control group. Analysis of apoptosis-related genes in oocytes after IVM revealed lesser transcript abundances for Bax gene, and higher transcript abundances for Bcl-2 gene in ALA-treated oocytes as compared with the control oocytes. In term of cleavage rate (considered as 2-cell progression), we did not observe any differences among the groups. However, ALA-100 group promoted more blastocyst 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 blastocyst formation and this might be due to the altered abundance of apoptosis-regulatory genes.

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

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 certain molecular markers. METHODS: Before brilliant cresyl blue (BCB) staining test, oocytes were classified as high, middle, and low cytoplasmic lipid content (HCLC, MCLC, and LCLC) and after the test as having low or high G6PDH-activity (BCB(+) and BCB(-), respectively). After maturation in vitro, a group of oocytes were subjected to IVF followed by in vitro embryo culture and another group was used for evaluation of expression of candidate genes. RESULTS: The cleavage and blastosyst rates were lowest (P < 0.05) in LCLC group, intermediate (P < 0.05) in MCLC group and highest (P < 0.05) in HCLC group. More (P < 0.05) oocytes in HCLC group were BCB(+), and higher (P < 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 was similar between BCB oocytes groups. However, the expression of ATP1A1 was higher (P < 0.05) for BCB(+) oocytes compared to BCB(-) oocytes. In addition, BAX transcript abundance was similar (P > 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 and objective predictor for subsequent developmental competence in vitro.

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

PURPOSE: 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. METHODS: Goat oocytes were exposed to brilliant cresyl blue (BCB) staining test and categorized into BCB(+) (blue-cytoplasm), and BCB(-) (colorless-cytoplasm) groups. A group of oocytes were not exposed to BCB test and was considered as a control group. After maturation in vitro, a group of oocytes were used for determination of nuclear status and intracellular GSH content while another group was subjected to parthenogenetic activation followed by in vitro embryo culture. RESULTS: We found that BCB(+) oocytes not only yielded higher rate of maturation, but also showed an increased level of intracellular GSH content than BCB(-) and control oocytes. Furthermore, BCB(+) oocytes produced more blastocysts than BCB(-) and control oocytes. Our data revealed that the expression of anti-apoptotic (Bcl-2) and pro-apoptotic (Bax) genes were interacted with G6PDH-activity in mature oocyte, their surrounding cumulus cells, and blastocyst-stage embryos. CONCLUSIONS: The results of this study demonstrate that selection of goat oocytes based on G6PDH-activity through the BCB test improves their developmental competence, increases intracellular GSH content, and affects the expression of the apoptosis-related genes.