mTOR is an essential gate in adapting the functional response of ovine trophoblast cells under stress-inducing environments.

INTRODUCTION: During the early stage of pregnancy trophoblast cells adapt to adverse uterine environments characterized by oxygen and nutrient deprivation. Autophagy is an intracellular degradation process that aims to promote cell survival in response to stressful conditions. Autophagy activation passes through the mechanistic target of rapamycin (mTOR), also known as a placental nutrient sensor. Here, we tested the hypothesis that ovine trophoblast cells may adapt to a suboptimal environment through an mTOR dependent regulation of cell survival with relevant implications for key placental functionality. METHODS: Primary ovine trophoblast cells subjected to mTOR inhibitor and low-nutrient conditions were used to explore how autophagy affects cellular functionality and expression of solute carriers' genes (SLCs). RESULTS: Autophagy activation was confirmed both in rapamycin-treated and low-nutrient conditions, through the detection of specific autophagic markers. However, p-mTOR activation seems to be severely modified only following rapamycin treatment whereas 24h of starvation allowed p-mTOR reactivation. Starvation promoted migration compared to normal culture conditions whereas all trophoblast functional activities were decreased in rapamycin treatment. Interestingly in both conditions, the autophagy-activated environment did not affect the progesterone release. mRNA expression of amino acid transporters remains largely undisturbed except for SLC43A2 and SLC38A4 which are downregulated in starved and rapamycin-treated cells, respectively. DISCUSSION: The study demonstrates that sheep trophoblast cells can adapt to adverse conditions in the early stage of placentation by balancing, in an mTOR dependent manner, nutrient recycling and transport with relevant effects for in vitro functional properties, which could potentially impact conceptus development and survival.

Exogenous melatonin ameliorates embryo-maternal cross-talk in early pregnancy in sheep.

In brief: Melatonin plays a crucial role in enhancing reproductive performance in small ruminants. This paper reveals the effects of exogenous melatonin on the placental and endometrial rearrangement in early pregnancy in sheep. Abstract: Early pregnancy losses cause 25% of pregnancy failures in small ruminants because of asynchrony between conceptus and uterine signals. In this context, melatonin plays a crucial role in sheep reproductive dynamics, but little is known about its effects during the peri-implantation period. We hypothesized that melatonin supports embryo implantation by modulating the uterine microenvironment. This study aimed to assess the effects of exogenous melatonin on the endometrial and early placental rearrangement. Ten multiparous ewes either did (MEL, n = 5) or did not (CTR, n = 5) receive a subcutaneous melatonin implant (18 mg) 50 days before a synchronized mating. On day 21 of pregnancy, the sheep were euthanized. MEL ewes exhibited a higher prolificity rate (2.8 vs 2.0 embryos/ewe) and plasma progesterone levels (3.84 vs 2.96 ng/mL, P < 0.05) than did CTR ewes. Groups did not differ significantly in embryo crown-rump length. MEL placentas had significantly (P < 0.001) more binucleated trophoblast cells in the chorion region, and ovine placental lactogen expression was significantly (P < 0.05) more strongly upregulated than in CTR. Exogenous melatonin increased significantly (P < 0.05) gene expression of angiogenic factors (VEGFA, VEGFR1, IGF1R), IFNAR2, and PR in the caruncular endometrium. Expression of the MT2 receptor in the endometrium and placenta was significantly (P < 0.05) higher in the MEL group. These results indicate that melatonin implants acted differentially on uterine and placental rearrangement. Melatonin increases differentiation in the placenta and induces changes that could promote vessel maturation in the endometrium, suggesting that it enhances the uterine microenvironment in the early stage of pregnancy in sheep.

A Deep Learning Approach for Accurate Path Loss Prediction in LoRaWAN Livestock Monitoring.

The agricultural sector is amidst an industrial revolution driven by the integration of sensing, communication, and artificial intelligence (AI). Within this context, the internet of things (IoT) takes center stage, particularly in facilitating remote livestock monitoring. Challenges persist, particularly in effective field communication, adequate coverage, and long-range data transmission. This study focuses on employing LoRa communication for livestock monitoring in mountainous pastures in the north-western Alps in Italy. The empirical assessment tackles the complexity of predicting LoRa path loss attributed to diverse land-cover types, highlighting the subtle difficulty of gateway deployment to ensure reliable coverage in real-world scenarios. Moreover, the high expense of densely deploying end devices makes it difficult to fully analyze LoRa link behavior, hindering a complete understanding of networking coverage in mountainous environments. This study aims to elucidate the stability of LoRa link performance in spatial dimensions and ascertain the extent of reliable communication coverage achievable by gateways in mountainous environments. Additionally, an innovative deep learning approach was proposed to accurately estimate path loss across challenging terrains. Remote sensing contributes to land-cover recognition, while Bidirectional Long Short-Term Memory (Bi-LSTM) enhances the path loss model's precision. Through rigorous implementation and comprehensive evaluation using collected experimental data, this deep learning approach significantly curtails estimation errors, outperforming established models. Our results demonstrate that our prediction model outperforms established models with a reduction in estimation error to less than 5 dB, marking a 2X improvement over state-of-the-art models. Overall, this study signifies a substantial advancement in IoT-driven livestock monitoring, presenting robust communication and precise path loss prediction in rugged landscapes.

Bovine Skeletal Muscle Satellite Cells: Isolation, Growth, and Differentiation.

Skeletal muscle in cattle occupies a large part of the animal's body mass and develops into an important source of nutrients for human nutrition. Recently, the attention on bovine myogenic cells is increased to develop strategies of cultured in vitro meat as an alternative food source, more sustainable, ethical, and healthy than traditional meat production. At present, investigating the proliferation and differentiation of bovine skeletal muscle myogenic cells in vitro maintains its importance in the study of the mechanisms underlying the physiological and pathological events affecting the skeletal muscle, but it is of particular interest in animal husbandry and the food industry fields.In cell-based biological research, cell lines are one of the favored experimental tools because a population of cells could proliferate indefinitely in vitro under different stimuli, but they are limited to addressing the relevant biological properties of a cell population. On the other hand, primary cells from normal animal tissues undergo a limited number of divisions in vitro before they enter senescence but preserve their original characteristics and functions, and researchers can acquire the opportunity to study the individual donors and not just cells.In this chapter, we provide a basic protocol to isolate satellite cells from the skeletal muscle of cattle to obtain a good number of myogenic cells that can grow in in vitro conditions and undergo multiple rounds of cell division (myoblasts) before entering differentiation (myotubes). Furthermore, the robust expansion of these cells leads to the possibility to investigate physiological events or disorders related to the skeletal muscle tissue.

Ovine Trophoblast Cells: Cell Isolation and Culturing from the Placenta at the Early Stage of Pregnancy.

Embryo development is dependent upon the exchange of oxygen and nutrients through the placenta, mainly composed of peculiar epithelioid cells, known as trophoblast cells. Normal trophoblast functionality plays a key role during the whole pregnancy, especially in the first stage of placentation. This chapter explains the techniques to obtain sheep primary trophoblast cells from the early placenta. Overall, procedures for cell isolation, culture, characterization, and cryopreservation are described.

Adaptation Response in Sheep: Ewes in Different Cortisol Clusters Reveal Changes in the Expression of Salivary miRNAs.

Farm procedures have an impact on animal welfare by activating the hypothalamic-pituitary-adrenal axis that induces a wide array of physiological responses. This adaptive system guarantees that the animal copes with environmental variations and it induces metabolic and molecular changes that can be quantified. MicroRNAs (miRNAs) play a key role in the regulation of homeostasis and emerging evidence has identified circulating miRNAs as promising biomarkers of stress-related disorders in animals. Based on a clustering analysis of salivary cortisol trends and levels, 20 ewes were classified into two different clusters. The introduction of a ram in the flock was identified as a common farm practice and reference time point to collect saliva samples. Sixteen miRNAs related to the adaptation response were selected. Among them, miR-16b, miR-21, miR-24, miR-26a, miR-27a, miR-99a, and miR-223 were amplified in saliva samples. Cluster 1 was characterized by a lower expression of miR-16b and miR-21 compared with Cluster 2 (p < 0.05). This study identified for the first time several miRNAs expressed in sheep saliva, pointing out significant differences in the expression patterns between the cortisol clusters. In addition, the trend analyses of these miRNAs resulted in clusters (p = 0.017), suggesting the possible cooperation of miR-16b and -21 in the integrated stress responses, as already demonstrated in other species as well. Other research to define the role of these miRNAs is needed, but the evaluation of the salivary miRNAs could support the selection of ewes for different profiles of response to sources of stressors common in the farm scenario.

Effects of Saccharomyces boulardii Supplementation on Nutritional Status, Fecal Parameters, Microbiota, and Mycobiota in Breeding Adult Dogs.

The aim of this study was to evaluate the effect of the administration of Saccharomyces boulardii on the nutritional, immunological, inflammatory, and stress status and on the composition of the gut microbiota and mycobiota in healthy adult dogs. A total of 25 American Staffordshire Terrier dogs were selected and randomly assigned to two groups: control (CTR, n = 12) and treated (TRT, n = 13) groups. No significant differences were found between the two groups regarding body weight, body condition score, and fecal score. No significant differences in microbiota/mycobiota, short chain fatty acids, indole/skatole, histamine, zonulin, or lactoferrin were detected. Indeed, supplementation with S. boulardii significantly decreased fecal calprotectin Immunoglobulin A, indicating an improvement in the gut well-being. Interestingly, fecal cortisol significantly decreased in dogs belonging to the TRT group compared to the CTR, suggesting both an improvement of the intestinal status and a reduction of stress, a common condition affecting animals managed in a breeding environment.

Circulating skeletal muscle related microRNAs profile in Piedmontese cattle during different age.

Piedmontese cattle is known for double-muscle phenotype. MicroRNAs (miRNAs) play important role as regulators in skeletal muscle physiological processes, and we hypothesize that plasma miRNAs expression profiles could be affected by skeletal muscle growth status related to age. Plasma samples of cattle were collected during four different ages from first week of life until the time of commercial end of the fattening period before slaughter. Small-RNA sequencing data analysis revealed the presence of 40% of muscle-related miRNAs among the top 25 highly expressed miRNAs and, 19 miRNAs showed differential expression too. Using qRT-PCR, we validated in a larger bovine population, miRNAs involved in skeletal muscle physiology pathways. Comparing new-born with the other age groups, miR-10b, miR-126-5p, miR-143 and miR-146b were significantly up-regulated, whereas miR-21-5p, miR-221, miR-223 and miR-30b-5p were significantly down-regulated. High expression levels of miR-23a in all the groups were found. Myostatin, a negative regulator of skeletal muscle hypertrophy, was predicted as the target gene for miR-23a and miR-126-5p and we demonstrated their direct binding. Correlation analysis revealed association between miRNAs expression profiles and animals' weights along the age. Circulating miRNAs could be promising for future studies on their biomarker potentialities to beef cattle selection.

Effect of sugar metabolite methylglyoxal on equine lamellar explants: An ex vivo model of laminitis.

Laminitis is one of the most devastating diseases in equine medicine, and although several etiopathogenetic mechanisms have been proposed, few clear answers have been identified to date. Several lines of evidence point towards its underlying pathology as being metabolism-related. In the carbonyl stress pathway, sugars are converted to methylglyoxal (MG)-a highly reactive α-oxoaldehyde, mainly derived during glycolysis in eukaryotic cells from the triose phosphates: D-glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. One common hypothesis is that MG could be synthesized during the digestive process in horses, and excessive levels absorbed into peripheral blood could be delivered to the foot and lead to alterations in the hoof lamellar structure. In the present study, employing an ex vivo experimental design, different concentrations of MG were applied to hoof explants (HE), which were then incubated and maintained in a specific medium for 24 and 48 h. Macroscopic and histological analyses and a separation force test were performed at 24 and 48 h post-MG application. Gene expression levels of matrix metalloproteinase (MMP)-2 and -14 and tissue inhibitor of metalloproteinase (TIMP)-2 were also measured at each time point for all experimental conditions. High concentrations of MG induced macroscopic and histological changes mimicking laminitis. The separation force test revealed that hoof tissue samples incubated for 24 h in a high concentration of MG, or with lower doses but for a longer period (48 h), demonstrated significant weaknesses, and samples were easily separated. All results support that high levels of MG could induce irreversible damage in HEs, mimicking laminitis in an ex vivo model.

External and internal EGFR-activating signals drive mammary epithelial cells proliferation and viability.

During puberty, the mammary gland undergoes an intense growth, dependent on the interplay between the Epidermal Growth Factor Receptor (EGFR) in the stroma and different mammary epithelial receptors. We hypothesize that EGFR expressed in the mammary epithelium also has a role in puberty and the epithelial cells can self-sustain by EGFR-mediated autocrine signaling. We adopted mammary cell lines from different species, as in vitro model for the epithelium, and we observed that EGFR-signaling positively affects their survival and proliferation. Once deprived of external growth factors, mammary cells still showed strong Erk 1/2 phosphorylation, abolished upon EGFR inhibition, coupled with a further reduction in survival and proliferation. Based on gene expression analysis, three EGFR-ligands (AREG, EREG and HBEGF) are likely to mediate this autocrine signaling. In conclusion, internal EGFR-activating signals sustain mammary epithelial cell proliferation and survival in vitro.

A New Approach to LCA Evaluation of Lamb Meat Production in Two Different Breeding Systems in Northern Italy.

Lamb meat production provides vital landscape-management and ecosystem services; however, ruminant farming produces a considerable share of the world's greenhouse gas emissions. To measure and compare the advantages and disadvantages of the intensification of livestock farming, an integrative analysis was conducted in this study by combining environmental impact analysis and animal welfare assessment. This approach is the first of its kind and is the innovative aspect of this paper. The methodology of Life Cycle Assessment (LCA) entails the holistic analysis of various impact categories and the associated emission quantities of products, services, and resources over their life cycle, including resource extraction and processing, production processes, transport, usage, and the end of life. The outlines of LCA are standardized in DIN EN ISO 14040/14044. To assess the environmental impacts of the production of lamb meat in northern Italy, two case studies were undertaken using the LCA software GaBi. The analysis is based on primary data from two sheep-breeding systems (semi-extensive and semi-intensive in alpine and continental bioregions, respectively) combined with inventory data from the GaBi database and data from the literature. The assessment was conducted for the functional unit of 1 kg of lamb meat and focuses on the impact categories global warming potential, acidification potential, and eutrophication potential. For an overall evaluation of the supply chain, we have also considered a parameter indicating animal welfare, in keeping with consumer concerns, employing an analysis of chronic stress as shown by cortisol accumulation. The goal is to derive models and recommendations for an efficient, more sustainable use of resources without compromising animal welfare, meat quality, and competitiveness. The aim of this study is to provide a standard for individualized sustainability analyses for European lamb production systems in the future. From the LCA perspective, the more intensive case-study farm showed a lower impact in global impact factors and a higher impact in local impact categories in comparison with the more extensively run farm that was studied. From the animal welfare perspective, lower amounts of the stress hormone cortisol were found on the extensively managed case-study farm.

In vitro and in vivo effects of toceranib phosphate on canine osteosarcoma cell lines and xenograft orthotopic models.

Canine osteosarcoma (OSA) is the most common primary malignant bone tumour in dogs, and it has a high metastatic rate and poor prognosis. Toceranib phosphate (TOC; Palladia, Zoetis) is a veterinary tyrosine kinase inhibitor that selectively inhibits VEGFR-2, PDGFRs and c-Kit, but its efficacy is not yet fully understood in the treatment of canine OSA. Here, we evaluated the functional effects of TOC on six OSA cell lines by transwell, wound healing and colony formation assays. Subsequently, two cell lines (Wall and Penny) were selected and were inoculated in mice by intrafemoral injection to develop an orthotopic xenograft model of canine OSA. For each cell line, 30 mice were xenografted; half of them were used as controls, and the other half were treated with TOC at 40 mg/kg body weight for 20 days. TOC inhibited cell growth of all cell lines, but reduced invasion and migration was only observed in Penny and Wall cell lines. In mice engrafted with Penny cells and subjected to TOC treatment, decreased tumour growth was observed, and PDGFRs and c-Kit mRNA were downregulated. Immunohistochemical analyses demonstrated a significant reduction of Ki67 staining in treated mice when compared to controls. The results obtained here demonstrate that TOC is able to slightly inhibit cell growth in vitro, while its effect is evident only in a Penny cell xenograft model, in which TOC significantly reduced tumour size and the Ki67 index without modifying apoptosis markers.

MicroRNAs as Biomarkers for Animal Health and Welfare in Livestock.

MicroRNAs (miRNAs) are small and highly conserved non-coding RNA molecules that orchestrate a wide range of biological processes through the post-transcriptional regulation of gene expression. An intriguing aspect in identifying these molecules as biomarkers is derived from their role in cell-to-cell communication, their active secretion from cells into the extracellular environment, their high stability in body fluids, and their ease of collection. All these features confer on miRNAs the potential to become a non-invasive tool to score animal welfare. There is growing interest in the importance of miRNAs as biomarkers for assessing the welfare of livestock during metabolic, environmental, and management stress, particularly in ruminants, pigs, and poultry. This review provides an overview of the current knowledge regarding the potential use of tissue and/or circulating miRNAs as biomarkers for the assessment of the health and welfare status in these livestock species.

Mammary Stem Cells in Domestic Animals: The Role of ROS.

Reactive oxygen species (ROS) are produced as a natural byproduct of the normal metabolism of oxygen and play significant roles in cell signaling and homeostasis. Although ROS have been involved in pathological processes as diverse as cancer, cardiovascular disease, and aging, they may to exert an effect even in a physiological context. In the central nervous system, stem cells and hematopoietic stem cells are early progenitors that contain lower levels of ROS than their more mature progeny. These different concentrations have been reported to be crucial for maintaining stem cell function. Mammary gland remodeling has been proposed to be organized through the activation and regulation of cells with stemness, either considered real stem cells or primitive precursors. Given the state of oxidative stress in the mammary gland tissue induced by high milk production, in particular in highly productive dairy cows; several studies have focused on the relationship between adult mammary stem cells and the oxidative state of the gland. The oxidative state of the mammary gland appears to be involved in the initial development and metastasis of breast cancer through interference with mammary cancerous stem cells. This review summarizes some links between the mammary stem and oxidative state of the gland.

Bovine Mammary Organoids: A Model to Study Epithelial Mammary Cells.

Bovine mammary organoids are cell aggregates that are produced by an association of a mechanical and an enzymatic dissociation of mammary gland tissue. They provide a useful source to isolate mammary epithelial cells, but can also be frozen as an intermediate dissociation step.Due to the strong cell-cell interactions among epithelial cells, the production and isolation of organoids is an efficient way to remove unwanted cell population of non-epithelial origin like fibroblasts.

Murine and Human Mammary Cancer Cell Lines: Functional Tests.

The biological characterization of mammary cancer cells is a prerequisite that helps the scientist understand some aspect of tumor biology. Once isolated from the tumor, cells are subjected to multiple tests that dissect their ability to growth, migrate, degrade the surrounding stroma, produce 3-dimensional structures and differentiate. Targeted inhibitors, when added to these tests, are used to unravel how specific growth factors, receptors, and intracellular translational pathways promote the ability of mammary tumor cells to achieve their biological behavior. Herein we describe a set of techniques used to put in focus the biological capacities in mammary cancer cells. When the characterization of a biological trait (e.g., proliferation) is assessable by multiple assays, we will limit the description to only one technique, possibly the easier to manage and that requires minimal laboratory equipment.

CD49f+ mammary epithelial cells decrease in milk from dairy cows stressed by overstocking during the dry period.

The work reported in this Research Communication describes the modification in epithelial cell populations during the first and the last month of milking in Holstein Friesian cows that have undergone different management during the dry period, and we report the differential expression of CD49f+ and cytokeratin18+ cell subpopulations. Twenty six cows were randomly divided into 2 balanced groups that were housed at stocking density of either 11 m2 (CTR) or 5 m2 from 21 ± 3 d before the expected calving until calving. Cells collected from milk samples taken in early lactation and late lactation were directly analysed for CD45, CD49f, cytokeratin 14, cytokeratin 18 and cell viability. We observed a differential expression with a significant reduction in CD49f+ (P < 0·01) and cytokeratin 18+ (P < 0·05) cells in early lactation. Differences were still evident in late lactation but were not significant. These observations suggest that mammary epithelial cell immunophenotypes could be associated with different animal management in the dry period and we hypothesise they may have a role as biomarkers for mammary gland function in dairy cows.

Deep Sequencing Reveals a Novel miR-22 Regulatory Network with Therapeutic Potential in Rhabdomyosarcoma.

Current therapeutic options for the pediatric cancer rhabdomyosarcoma have not improved significantly, especially for metastatic rhabdomyosarcoma. In the current work, we performed a deep miRNA profiling of the three major human rhabdomyosarcoma subtypes, along with cell lines and normal muscle, to identify novel molecular circuits with therapeutic potential. The signature we determined could discriminate rhabdomyosarcoma from muscle, revealing a subset of muscle-enriched miRNA (myomiR), including miR-22, which was strongly underexpressed in tumors. miR-22 was physiologically induced during normal myogenic differentiation and was transcriptionally regulated by MyoD, confirming its identity as a myomiR. Once introduced into rhabdomyosarcoma cells, miR-22 decreased cell proliferation, anchorage-independent growth, invasiveness, and promoted apoptosis. Moreover, restoring miR-22 expression blocked tumor growth and prevented tumor dissemination in vivo Gene expression profiling analysis of miR-22-expressing cells suggested TACC1 and RAB5B as possible direct miR-22 targets. Accordingly, loss- and gain-of-function experiments defined the biological relevance of these genes in rhabdomyosarcoma pathogenesis. Finally, we demonstrated the ability of miR-22 to intercept and overcome the intrinsic resistance to MEK inhibition based on ERBB3 upregulation. Overall, our results identified a novel miR-22 regulatory network with critical therapeutic implications in rhabdomyosarcoma. Cancer Res; 76(20); 6095-106. ©2016 AACR.

Hepatocyte Growth Factor-mediated satellite cells niche perturbation promotes development of distinct sarcoma subtypes.

Embryonal Rhabdomyosarcoma (ERMS) and Undifferentiated Pleomorphic Sarcoma (UPS) are distinct sarcoma subtypes. Here we investigate the relevance of the satellite cell (SC) niche in sarcoma development by using Hepatocyte Growth Factor (HGF) to perturb the niche microenvironment. In a Pax7 wild type background, HGF stimulation mainly causes ERMS that originate from satellite cells following a process of multistep progression. Conversely, in a Pax7 null genotype ERMS incidence drops, while UPS becomes the most frequent subtype. Murine EfRMS display genetic heterogeneity similar to their human counterpart. Altogether, our data demonstrate that selective perturbation of the SC niche results in distinct sarcoma subtypes in a Pax7 lineage-dependent manner, and define a critical role for the Met axis in sarcoma initiation. Finally, our results provide a rationale for the use of combination therapy, tailored on specific amplifications and activated signaling pathways, to minimize resistance emerging from sarcomas heterogeneity.

Bovine CD49 positive-cell subpopulation remarkably increases in mammary epithelial cells that retain a stem-like phenotype.

We previously proved that adult stem cells reside in the bovine mammary gland and possess an intrinsic potential to generate a functional mammary outgrowth. The aim of this study was to investigate on the immunophenotyping features retained by mammary stem-like cells detected in long term culture. Flow cytometry analysis showed different subpopulations of mammary epithelial cells emerging according to the timing of cell culture. CD49f(+)-cells significantly increased during the culture (p<0.01) and a similar trend was observed, even if less regular, for CD29(+) and ALDH1 positive cell populations. No difference during the culture was observed for CD24 positive cells but after 35 days of culture a subset of cells, CD49f positive, still retained regenerative capabilities in in vivo xenotransplants. These cells were able to form organized pseudo-alveoli when transplanted in immunodeficient mice. These results prove the presence of a multipotent cell subpopulation that retain a strong epithelial induction, confirmed in in vivo xenotransplants with a presumable in vitro expansion of the primitive population of adult mammary stem cells.