The presence of an embryo affects day 14 uterine transcriptome depending on the nutritional status in sheep. b. Immune system and uterine remodeling.

Transcriptomics and bioinformatics were used to investigate the potential interactions of undernutrition and the presence of the conceptus at the time of maternal recognition of pregnancy on uterine immune system and remodeling. Adult Rasa Aragonesa ewes were allocated to one of two planes of nutrition for 28 days: maintenance energy intake (control; 5 cyclic, 6 pregnant ewes) providing 7.8 MJ of metabolisable energy and 0.5 maintenance intake (undernourished; 6 cyclic, 7 pregnant ewes) providing 3.9 MJ of metabolisable energy per ewe. Uterine gene expression was measured using Agilent 15 K Sheep Microarray chip on day 14 of estrus or pregnancy. Functional bioinformatics analyses were performed using PANTHER (Protein ANalysis THrough Evolutionary Relationships) Classification System. Pregnancy affected the expression of 18 genes in both control and undernourished ewes, underscoring the relevance for embryo-maternal interactions. Immune system evidenced by classical interferon stimulated genes were activated in control and -in a lesser extent-in undernourished pregnant vs cyclic ewes. Genes involved in uterine remodeling such as protein metabolism were also upregulated with the presence of an embryo in control and undernourished ewes. However, relevant genes for the adaptation of the uterus to the embryo were differentially expressed between pregnant vs cyclic ewes both in control and undernourished groups. Undernutrition alone led to an overall weak activation of immune system pathways both in cyclic and pregnant ewes. Data revealed that cellular and immune adaptations of the uterus to pregnancy are dependent on the nutritional status.

The embryo affects day 14 uterine transcriptome depending on nutritional status in sheep. a. Metabolic adaptation to pregnancy in nourished and undernourished ewes.

This study investigated the effects of undernutrition and the presence of the conceptus at the time of maternal recognition of pregnancy on the expression of uterine indicators of metabolism in ewes. Adult Rasa Aragonesa ewes were allocated to one of two planes of nutrition for 28 days: maintenance energy intake (control; 5 cyclic and 6 pregnant ewes) providing 7.8 MJ of metabolisable energy, and 0.5 maintenance intake (undernourished; 6 cyclic and 7 pregnant ewes) providing 3.9 MJ of metabolisable energy per ewe. RNA from intercaruncular uterine tissue was harvested at slaughter on Day 14 of estrous cycle or pregnancy, and hybridized to the Agilent 15K Sheep Microarray chip. Functional bioinformatics analyses were performed using PANTHER (Protein ANalysis THrough Evolutionary Relationships) Classification System. The presence of the embryo upregulated expression of genes encoding peptide and monocarboxylate transporters regardless of nutritional treatment, although the degree of gene expression was lower in undernourished ewes. Genes encoding enzymes involved in glycolysis were downregulated both in pregnant control and undernourished ewes, probably as a compensatory mechanism for the increased glucose transport to the uterus. Compared with control cyclic ewes, control pregnant ewes had greater expression of genes involved in oxidation of fatty acids, suggesting increased uterine energy demands. This was not observed in undernourished pregnant animals when compared to undernourished cyclic ewes; nevertheless, those animals had lower uterine expression of enzymes involved in fatty acid biosynthesis. The presence of the embryo upregulated genes involved in electron transport probably as a result of increased energy demands for pregnancy. Overall, the data indicate that depending on the nutritional status of ewe, pregnancy alters gene expression of metabolic pathways related to energy generation in the uterus. An impairment in nutrient transport and metabolism in the uterus of pregnant undernourished ewes may explain the greater embryo mortality associated with undernutrition.

Innate immune responses induced by lipopolysaccharide and lipoteichoic acid in primary goat mammary epithelial cells.

BACKGROUND: Innate immune responses induced by in vitro stimulation of primary mammary epithelial cells (MEC) using Gram-negative lipopolysaccharide (LPS) and Gram-positive lipoteichoic acid (LTA) bacterial cell wall components are well- characterized in bovine species. The objective of the current study was to characterize the downstream regulation of the inflammatory response induced by Toll-like receptors in primary goat MEC (pgMEC). We performed quantitative real-time RT-PCR (qPCR) to measure mRNA levels of 9 genes involved in transcriptional regulation or antibacterial activity: Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), prostaglandin-endoperoxide synthase 2 (PTGS2), interferon induced protein with tetratricopeptide repeats 3 (IFIT3), interferon regulatory factor 3 (IRF3), myeloid differentiation primary response 88 (MYD88), nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NFKB1), Toll interacting protein (TOLLIP), and lactoferrin (LTF). Furthermore, we analyzed 7 cytokines involved in Toll-like receptor signaling pathways: C-C motif chemokine ligand 2 (CCL2), C-C motif chemokine ligand 5 (CCL5), C-X-C motif chemokine ligand 6 (CXCL6), interleukin 8 (CXCL8), interleukin 1 beta (IL1B), interleukin 6 (IL6), and tumor necrosis factor alpha (TNF). RESULTS: Stimulation of pgMEC with LPS for 3 h led to an increase in expression of CCL2, CXCL6, IL6, CXCL8, PTGS2, IFIT3, MYD88, NFKB1, and TLR4 (P < 0.05). Except for IL6, and PTGS2, the same genes had greater expression than controls at 6 h post-LPS (P < 0.05). Expression of CCL5, PTGS2, IFIT3, NFKB1, TLR4, and TOLLIP was greater than controls after 3 h of incubation with LTA (P < 0.05). Compared to controls, stimulation with LTA for 6 h led to greater expression of PTGS2, IFIT3, NFKB1, and TOLLIP (P < 0.05) whereas the expression of CXCL6, CXCL8, and TLR4 was lower (P < 0.05). At 3 h incubation with both toxins compared to controls a greater expression (P < 0.05) of CCL2, CCL5, CXCL6, CXCL8, IL6, PTGS2, IFIT3, IRF3, MYD88, and NFKB1 was detected. After 6 h of incubation with both toxins, the expression of CCL2, CXCL6, IFIT3, MYD88, NFKB1, and TLR4 was higher than the controls (P < 0.05). CONCLUSIONS: Data indicate that in the goat MEC, LTA induces a weaker inflammatory response than LPS. This may be related to the observation that gram-positive bacteria cause chronic mastitis more often than gram-negative infections.

Detecting ß-Casein Variation in Bovine Milk.

In bovine species, ß-casein (ß-CN) is characterized by genetic polymorphism. The two most common protein variants are ß-CN A² (the original one) and A¹, differing from A² for one amino acid substitution (Pro67 to His67). Several bioactive peptides affecting milk nutritional properties can originate from ß-CN. Among them, ß-casomorphin-7 (BCM7) ranging from amino acid 60 to 66 can be released more easily from ß-CN variants carrying His67 (A¹ type) instead of Pro67 (A² type). Nowadays, "A2 milk" is produced in different countries claiming its potential benefits in human health. The aim of this study was to further develop and apply an isoelectric focusing electrophoresis (IEF) method to bulk and individual milk samples in order to improve its use for ß-CN studies. We succeeded in identifying A2 milk samples correctly and quantifying the percentage of A², A¹, and B variants in bulk samples not derived from A2 milk as well as in individual milk samples. The method allows us to quantify the relative proportion of ß-CN variants in whole milk without eliminating whey protein by acid or enzymatic precipitation of caseins. The aim of this study was also to study the different behavior of ß-CN and ß-lactoglobulin (ß-LG) in the presence of trichloroacetic acid (TCA). The higher sensitivity of ß-CN to TCA allows quantifying ß-CN variants after TCA fixation because ß-LG is not visible. Monitoring ß-CN variation in cattle breeds is important in order to maintain a certain balance between Pro67 and His67 in dairy products. Overall, the debate between A1 and A2 milk needs further investigation.

Variation of vitamin D in cow's milk and interaction with ß-lactoglobulin.

Vitamin D is the collective name for a group of closely related lipids, whose main biological function is to maintain serum calcium and phosphorus concentrations within the normal range by enhancing the efficiency of the small intestine to absorb these minerals from the diet. We used a commercially available ELISA method for the determination of vitamin D in bovine milk. Individual milk samples from two different Italian Friesian herds were analysed. The enzyme immunoassay method used was confirmed as a useful tool to measure the vitamin D in the milk as it greatly reduces the time required to perform the conventional HPLC analysis. An interesting variation was found among individual animals that may be associated with management factors and specific genetic effects. A relationship was highlighted between vitamin D and the genetic polymorphism of ß-lactoglobulin, the main bovine whey protein which is involved in the transport of small hydrophobic molecules such as retinol and vitamin D. The relatively high content of vitamin D in most milk samples suggests an opportunity to improve the natural content of vitamin D in milk either by acting on the herd management or selecting individuals genetically predisposed to produce milk with a higher vitamin D content.

Direct effects of casein phosphopeptides on growth and differentiation of in vitro cultured osteoblastic cells (MC3T3-E1).

Casein phosphopeptides (CPPs) obtained by enzymatic hydrolysis in vitro of caseins, have been shown to enhance calcium solubility and to increase the calcification of embryonic rat bones in their diaphyseal area. Little is known about the direct effects of CPPs on cultured osteoblastic cells. Calcium in the microenvironment surrounding bone cells is not only important for the mineralization of the extracellular matrix, but it is believed to provide preosteblasts with a signal that modulates their proliferation and differentiation. The aim of the present study was to investigate the direct effects of four selected casein phosphopeptides on osteoblastic cell (MC3T3-E1 cells) viability and differentiation. The selected peptides have been obtained by chemical synthesis and differed in the number of phosphorylated sites and in the amino acid spacing out two phosphorylated sites, in order to further characterize the relationship between structure and function. The results obtained in this work demonstrated that CPPs may directly affect osteoblast-like cell growth, calcium uptake and ultimately calcium deposition in the extracellular matrix. The effects exerted by distinct CPPs on osteogenesis in vitro can be either stimulatory or inhibitory. Differential short amino acid sequences in their molecules, like the -SpEE- and the -SpTSpEE-motifs, are likely the molecular determinants for their biological activities on osteoblastic cells. Moreover, two genetic variants of CPPs showing one amino acid change in their sequence may profoundly differ in their biological activities. Finally, our data may also suggest important clues about the role of intrinsic phosphorylated peptides derived from endogenous phosphorylated proteins in bone metabolism, apart from extrinsic CPPs.