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During bovine mastitis, immune responses include the release of cytokines and the recruitment of leukocytes, resulting in profound structural and functional changes in the mammary gland. Our aims were to delineate systemic and local cytokine responses and to quantify histological changes in the mammary tissue of lactating cows after acute intramammary lipopolysaccharide (LPS) challenge. Ten multiparous dairy cows were paired to either treatment (TRT) or control (CON) groups. For TRT cows, one side of the udder was randomly assigned to receive treatment with LPS (50 µg in 10 mL of saline, TL) into both the front and rear quarters; the contralateral quarters received saline (10 mL). Udder-halves of CON cows were similarly assigned randomly to receive either saline (10 mL, CS) or no infusion (untreated). Temporal changes in the concentrations of 15 cytokines in the blood (0, 3, 6, 12, and 24 h relative to the LPS infusion) and in mammary tissue (0, 3, and 12 h) were determined, as were concomitant changes in mammary histology. The cytokines IL-6, IL-10, MCP-1, and MIP-1ß showed a systemic response as their concentrations were significantly different in the plasma of TRT cows as compared with CON cows after LPS challenge. The cytokines IL-1α, IL-1ß, IL-6, IL-8, IL-17A, IL-36RA, IP-10, MCP-1, MIP-1α, MIP-1ß, TNF-α, and VEGF-A showed a local response in TL glands, and 8 cytokines, IL-1ß, IL-6, IL-10, IL-17A, IL-36RA, IP-10, MIP-1ß, and VEGF-A showed systemic changes in the nonchallenged mammary glands adjacent to LPS-infused glands. Endotoxin challenge evoked changes in the histology of mammary tissue that included a 5.2- and 7.2-fold increases in the number of neutrophils in alveolar lumens at 3 h and 12 h, respectively. In summary, LPS challenge induced specific local and systemic responses in cytokine induction and elicited neutrophil infiltration in bovine mammary tissue.
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Enfermedades de los Bovinos , Mastitis Bovina , Femenino , Bovinos , Animales , Citocinas/análisis , Lipopolisacáridos/farmacología , Lipopolisacáridos/análisis , Lactancia , Interleucina-10 , Leche/química , Interleucina-17/análisis , Quimiocina CCL4/análisis , Quimiocina CXCL10/análisis , Interleucina-6 , Factor A de Crecimiento Endotelial Vascular , Glándulas Mamarias AnimalesRESUMEN
This study aimed to investigate and characterize the spermatogonial stem cells (SSCs) in buffaloes at different stages of development, including prenatal, neonatal, prepubertal, and adult testes. We sought a comprehensive understanding of these cells through a combination of histological, immunohistochemical, and ultrastructural analyses. Specifically, we examined changes in the expression of two potential SSC markers, OCT4 and PGP9.5, using immunohistochemistry. Additionally, we conducted a real-time quantitative polymerase chain reaction (RT-qPCR) to assess the relative gene expression of OCT4 and PGP9.5. The relative expression of the OCT4 gene was down-regulated in the adult testes compared to its expression during prepubertal and neonatal life. The relative expression of the PGP9.5 gene was up-regulated in the neonatal testes and down-regulated in the prepubertal and adult testes. The spermatogonia were round, oval-to-ellipsoidal cells lying over the basement membrane (BM) with a round-to-oval nucleus. Based on the immunoexpression of the putative SSC markers, OCT4 and PGP9.5, we concluded that the proportion of stem cells was highest during the neonatal stage, followed by the prepubertal and prenatal stages. This finding sheds light on the dynamics of spermatogonial stem cells in buffalo testes at different developmental stages, providing valuable insights into these cells' regulation and potential applications.
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Búfalos , Testículo , Masculino , Animales , Testículo/metabolismo , Búfalos/genética , Espermatogonias/metabolismo , Células Cultivadas , Expresión GénicaRESUMEN
The utility of animal stem cells finds implications in enhancing milk, meat, and fiber production and serving animal models for human diseases. Stem cells are involved in tissue development, growth, and repair, and in regenerative therapy. Caprine embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and other tissue-specific adult stem cells (ASCs) have tremendous potential for their use in regenerative medicine. The application of goat ESCs, iPSCs, mammary stem cells (MaSC), mesenchymal stem cells (MSCs), spermatogonial stem cells (SSCs) and others can find their implication in increasing caprine production potential and human disease model. The onset of the disease and therapeutic effects of stem cells of many human diseases like sub-fertility, joint conditions, intervertebral disc defects, osteoarthritis, and chondrogenesis can be well studied in goats. Increasing evidence of MSCs and their secreted factors have drawn the attention of animal scientists in regenerative medicine. This review summarizes a comprehensive overview of research made on caprine stem cells and illustrates some potential applications of stem cells in caprine regenerative medicine and their utility as a model animal in understanding human diseases.
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Células Madre Pluripotentes Inducidas , Células Madre Mesenquimatosas , Adulto , Animales , Humanos , Cabras , Células Madre Embrionarias , Medicina Regenerativa , Diferenciación CelularRESUMEN
Adult stem cells like mammary and mesenchymal stem cells have received significant attention because these stem cells possess therapeutic potential in treating many animal diseases. These cells can be administered in an autologous or allogenic fashion, either freshly isolated from the donor tissue or previously cultured and expanded in vitro. The expansion of adult stem cells is a prerequisite before therapeutic application because sufficient numbers are required in dosage calculation. Stem cells directly and indirectly (by secreting various growth factors and angiogenic factors called secretome) act to repair and regenerate injured tissues. Recent studies on mammary stem cells showed in vivo and in vitro expansion ability by removing the blockage of asymmetrical cell division. Compounds like purine analogs (xanthosine, xanthine, and inosine) or hormones (progesterone and bST) help increase stem cell population by promoting cell division. Such methodology of enhancing stem cell number, either in vivo or in vitro, may help in preclinical studies for translational research like treating diseases such as mastitis. The application of mesenchymal stem cells has also been shown to benefit mammary gland health due to the 'homing' property of stem cells. In addition to that, the multiple positive effects of stem cell secretome are on mammary tissue; healing and killing bacteria is novel in the production of quality milk. This systematic review discusses some of the studies on stem cells that have been useful in increasing the stem cell population and increasing mammary stem/progenitor cells. Finally, we provide insights into how enhancing mammary stem cell population could potentially increase terminally differentiated cells, ultimately leading to more milk production.
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Células Madre Adultas , Leche , Animales , Diferenciación Celular , Femenino , Humanos , Glándulas Mamarias Animales/metabolismo , Leche/metabolismo , Células MadreRESUMEN
Livestock production contributes to a significant part of the economy in developing countries. Although artificial insemination techniques brought substantial improvements in reproductive efficiency, male infertility remains a leading challenge in livestock. Current strategies for the diagnosis of male infertility largely depend on the evaluation of semen parameters and fail to diagnose idiopathic infertility in most cases. Recent evidences show that spermatozoa contains a suit of RNA population whose profile differs between fertile and infertile males. Studies have also demonstrated the crucial roles of spermatozoal RNA (spRNA) in spermatogenesis, fertilization, and early embryonic development. Thus, the spRNA profile may serve as unique molecular signatures of fertile sperm and may play pivotal roles in the diagnosis and treatment of male fertility. This manuscript provides an update on various spRNA populations, including protein-coding and non-coding RNAs, in livestock species and their potential role in semen quality, particularly sperm motility, freezability, and fertility. The contribution of seminal plasma to the spRNA population is also discussed. Furthermore, we discussed the significance of rare non-coding RNAs (ncRNAs) such as long ncRNAs (lncRNAs) and circular RNAs (circRNAs) in spermatogenic events.
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Mammary stem cells (MaSC) are essential for growth and maintenance of mammary epithelium. Previous studies have utilized morphological characteristics or retention of bromodeoxyuridine (BrdU) label to identify MaSC and progenitor cells, these approaches may not be feasible or may not identify all resident stem cells. Alternatively, these special cells may be identified by assessing protein and mRNA expression of appropriate markers. The focus of this study was to assess the staining patterns and in situ quantification of novel candidate markers for bovine MaSC/progenitor cells. The candidate markers for MaSC/progenitor cells for immunohistochemical analysis were: NR5A2, NUP153, HNF4A, USP15 and FNDC3B and for in situ transcripts quantification were HNF4A and NUP153. We also evaluated protein expression pattern of presumptive MaSC markers known from the literature namely, ALDH1, MSI1 and Notch3. We found that NR5A2, NUP153, HNF4A and USP15-labeled cells represented 2.5-6% of epithelial cells prepubertally and were distributed in a fashion consistent with the location and abundance of MaSC/progenitor cells. A transient increase (10-37%) in expression of these markers was observed at peak lactation. FNDC3B was localized mainly in the nucleus prepubertally and in the cytoplasm of myoepithelial cells and nuclei of a limited number of alveolar cells during lactation. Abundant expression (~ 48%) and luminal localization of ALDH1 precludes its use as a bovine MaSC marker but may include transamplifying progenitor cells. MSI1 staining was consistent with MaSC localization. Onset of lumen formation in mammary ducts of prepubertal gland was associated with Notch 3 expression in the apical surface of luminal cells. RNAscope analysis of HNF4A and NUP153 transcripts in calf mammary gland showed very low copy numbers in a few epithelial cells, supporting the idea that these markers are expressed by fewer cells of epithelial origin. This study suggests that NR5A2, NUP153, HNF4A, USP15 and FNDC3B are likely markers for bovine MaSC/progenitor cells. Quantification of RNA transcripts of HNF4A and NUP153 in bovine MEC as potential MaSC markers are novel. Further studies to correlate protein expression of these markers with their transcripts level using single cell analysis in larger samples in lactating cow at different physiological stages are warranted.
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Biomarcadores/metabolismo , Fibronectinas/metabolismo , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Células Madre/citología , Células Madre/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Animales , Bovinos , Diferenciación Celular/fisiología , Línea Celular , FemeninoRESUMEN
BACKGROUND: MicroRNAs play key roles in host-pathogen-interactions and disease pathogenesis. Our aim was to characterize the differentially expressed miRNAs in the blood cells of diseased (Brucellosis-positive, Johne's disease-positive) and healthy- water buffaloes. The pooled small-RNA samples of each group were sequenced on Ion Torrent Personal Genome Machine (PGM) sequencer and the data were analyzed for differential expression. RESULTS: Here we identified 274 known miRNAs with bovine homologs and 36 novel mature-star miRNAs from the sequnces of small RNA libraries. Overall 195 miRNAs were common to all the three groups. Certain miRNAs such as bta-miR-21-5p, -26a, -29a/b, -30d - 103, - 140, - 150, - 191, - 374, - 1434-5p,-1260b, - 2484 and let-7 members were abundantly expressed in diseased groups. Bta-miR-1434-5p, - 188, -200c were up-regulated (> 1.5 folds) while bta-miR-27a-5p, -34b and -2285x were down-regulated (> 100 folds) in Brucellosis group. In Johne's Disease group, only 3 miRNAs (bta-miR-1434-5p, - 2340 and - 2484) were up-regulated (> 1.5 folds). The functional classification of miRNA target genes into gene ontology (GO) terms indicated their involvement in innate immunity and cellular process of disease pathogenesis. Expression profile of four differentially expressed miRNAs (bta-miR-9-5p, - 677, - 331-3p and - 2440) and eight predicted target-genes were validated through reverse transcriptase qPCR. CONCLUSION: This study provides a valuable frame of reference for elucidation of regulatory roles of miRNAs associated with disease pathogenesis in water buffaloes as well as identification of miRNA biomarkers for disease diagnosis and treatment.
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The aim of this study was to investigate the mechanisms underlying the reduced milk production during mastitis. We hypothesized that bacterial endotoxin induces hypoxia, oxidative stress, and cell apoptosis while inhibiting milk gene expression in the mammary gland. To test this hypothesis, the left and right sides of the 4th pair of mouse mammary glands were alternatively injected with either lipopolysaccharide (LPS, E. coli 055: B5, 100 µL of 0.2 mg/mL) or sterile PBS through the teat meatus 3 days postpartum. At 10.5 and 22.5 h postinjection, pimonidazole HCl, a hypoxyprobe, was injected intraperitoneally. At 12 or 24 h after the LPS injection, the 4th glands were individually collected (n = 8) and analyzed. LPS treatment induced mammary inflammation at both 12 and 24 h but promoted cell apoptosis only at 12 h. Consistently, H2O2 content was increased at 12 h (P < 0.01), but dropped dramatically at 24 h (P < 0.01) in the LPS-treated gland. Nevertheless, the total antioxidative capacity in tissue tended to be decreased by LPS at both 12 and 24 h (P = 0.07 and 0.06, respectively). In agreement with these findings, LPS increased or tended to increase the mRNA expression of antioxidative genes Nqo1 at 12 h (P = 0.05) and SLC7A11 at 24 h (P = 0.08). In addition, LPS inhibited mammary expression of Csn2 and Lalba across time and protein expression of Csn1s1 at 24 h (P < 0.05). Furthermore, hypoxyprobe staining intensity was greater in the alveoli of the PBS-treated gland than the LPS-treated gland at both 12 and 24 h, demonstrating a rise in oxygen tension by LPS treatment. In summary, our observations indicated that while intramammary LPS challenge incurs inflammation, it induces oxidative stress, increases cell apoptosis and oxygen tension, and differentially inhibits the milk protein expression in the mammary gland.
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Hipoxia de la Célula/fisiología , Endotoxinas/efectos adversos , Escherichia coli/patogenicidad , Glándulas Mamarias Animales/fisiología , Proteínas de la Leche/metabolismo , Estrés Oxidativo/fisiología , Animales , Femenino , RatonesRESUMEN
Aldehyde dehydrogenase 1 (ALDH1) and hepatocyte nuclear factor 4A (HNF4A) are the putative mammary stem cell markers. Tissue necrosis factor alpha (TNFA) is involved in inflammation-associated carcinogenesis and cell proliferation. In this study, the gene expression profile of ALDH1, HNF4A and TNFA of buffalo mammary tissue using real-time quantitative PCR (RT-qPCR). Analysis of RT-qPCR data revealed that the relative expression (log2 fold change) of ALDH1 and TNFA during mastitis (vs. lactation) was increased (P < .05) by 2.98 and 4.71, respectively. The relative expression (log2 fold change; -7.39) of stem cell marker, HNF4A was decreased (P < .05) during mastitis. Histological analysis of mammary tissue during mastitis showed thickening of stroma and occasionally hyperplasia, predominantly in prepubertal and non-lactating animals. Although, the level of expression of these genes may vary, depending upon the physiological stage of the animals, however expression of ALDH1 and TNFA was high during mastitis. A systematic study on large samples of buffalo mammary tissue with appropriate comparisons needs to be evaluated with these markers for prognosis of buffalo mammary health.
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Familia de Aldehído Deshidrogenasa 1/metabolismo , Factor Nuclear 4 del Hepatocito/metabolismo , Glándulas Mamarias Animales , Mastitis Bovina , Factor de Necrosis Tumoral alfa/metabolismo , Familia de Aldehído Deshidrogenasa 1/genética , Animales , Búfalos/genética , Búfalos/metabolismo , Bovinos , Factor Nuclear 4 del Hepatocito/genética , Glándulas Mamarias Animales/química , Glándulas Mamarias Animales/enzimología , Glándulas Mamarias Animales/metabolismo , Mastitis Bovina/enzimología , Mastitis Bovina/metabolismo , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The mammary gland undergoes distinct periods of growth, development, and secretory activity. During bovine lactation, a gradual decrease in the number of mammary epithelial cells largely accounts for the decline in milk production with advancing lactation. The net decline in cell number (approx. 50%) is due to cell death but is simultaneously accompanied by cell renewal. Although the rate of cell proliferation is slow, by the end of lactation most cells in the gland were formed after calving. Typically milking is terminated when cows are in the final 2 mo of pregnancy. This causes regenerative involution, wherein extensive cell replacement and mammary growth occurs. We hypothesized that replacement of senescent secretory cells and progenitor cells during the dry period increases milk yield in the next lactation. Analysis of global gene expression revealed networks and canonical pathways during regenerative involution that support cell turnover and mammary growth, and reflect oxidative stress, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress. Immune responses consistent with influx of neutrophils, macrophages, and lymphocytes, and processes that support mammary differentiation and lactogenesis were also evident. Data also suggest that replication of stem and progenitor cells occurs during the dry period. Relying on long-term retention of bromodeoxyuridine-labeled DNA, we identified putative bovine mammary stem cells. These label-retaining epithelial cells (LREC) are in low abundance within mammary epithelium (<1%), predominantly estrogen receptor-negative, and localized in a basal or suprabasal layer of the epithelium. Analyses of gene expression in laser-microdissected LREC are consistent with the concept that LREC represent stem cells and progenitor cells, which differ in properties and location within the epithelial layer. We identified potential markers for these cells and have increased their number by infusing xanthosine through the teat canal of prepubertal heifers. Altering population dynamics of mammary stem and progenitor cells during the mammary cycle may be a means to increase efficiency of milk production.
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Bovinos/fisiología , Leche/metabolismo , Dinámica Poblacional , Animales , Bromodesoxiuridina/química , Recuento de Células/veterinaria , Diferenciación Celular , Proliferación Celular , Células Epiteliales/metabolismo , Epitelio/metabolismo , Femenino , Lactancia , Glándulas Mamarias Animales/metabolismo , Embarazo , Ribonucleósidos/administración & dosificación , Células Madre/metabolismo , XantinasRESUMEN
Although water buffaloes are the main milk-producing animals in Indian subcontinent, only limited attempts have been made to identify canonical pathways and gene regulatory networks operating within the mammary glands of these animals. Such information is important for identifying unique transcriptome signatures in the mammary glands of diseased animals. In this report, we analyzed the transcription profile of 3 prepubertal buffalo mammary glands and identified common genes (mean FPKM > 0.2 in all samples) operating in the glands. Among 19,994 protein coding genes, 14,678 genes expressed and 5316 unique genes did not express in prepubertal buffalo mammary glands. Of these 14,678 expressed genes, 79% comprised a ubiquitous transcriptome that was dominated by very lowly expressed genes (51%). The percentage of rarely, moderately, and abundantly expressed genes was 25%, 2%, and 1%, respectively. Gene Ontology (GO) terms reflected in the expression of common genes (mean FPKM > 5.0) for molecular function were related to binding and catalytic activity. Products of these genes were involved in metabolic and cellular processes and belong to nucleic acid binding proteins. The canonical pathways for growth of mammary glands included integrin signaling, inflammation, GnRH and Wnt pathways. KEGG enriched pathways revealed many pathways of cancer including ribosome, splisosome, endocytosis, and ubiquitin-mediated proteolysis, pathways for viral infection, and bacterial invasion of epithelial. Highly expressed genes (mean FPKM > 500 included beta-actin (ACTB), beta-2 microglobulin (B2M), caseins (CSN2, CNS3), collagens (COL1A1, COL3A1), translation elongation factors (EEF1A1, EEF1G, EEF2), keratins (KRT15, KRT19), major histocompatibility complex genes (CD74, JSP.1), vimentin (VIM), and osteopontin (SPP1). Interestingly, expression of milk protein genes in prepubertal glands opens possible roles of these genes in development of mammary glands. We report the whole transcriptomic signature of prepubertal buffalo mammary gland and indicated its molecular signature is similar to cancer type.
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Búfalos/genética , Glándulas Mamarias Animales/metabolismo , Transcriptoma , Animales , Femenino , Perfilación de la Expresión Génica , Humanos , Glándulas Mamarias Animales/crecimiento & desarrolloRESUMEN
This study examined the hypothesis that xanthosine (XS) treatment would promote mammary-specific gene expression and stem cell transcripts and have a positive influence on milk yield of dairy goats. Seven primiparous Beetal goats were assigned to the study. Five days after kidding, one gland (either left or right) was infused with XS (TRT) twice daily for 3 d and the other gland with no XS infusion served as a control (CON). Mammary biopsies were collected at 10 d and RNA was isolated. Gene expression analysis of milk synthesis genes, mammary stem/progenitor cell markers, cell proliferation and differentiation markers were performed using real time quantitative PCR (RT-qPCR). Results showed that the transcripts of milk synthesis genes (BLG4, CSN2, LALBA, FABP3, CD36) and mammary stem/progenitor cell markers (ALDH1 and NR5A2) were increased in as a result of XS treatment. Average milk yield in TRT glands was increased marginally (approximately ~2% P = 0·05, paired t-test) per gland relative to CON gland until 7 wk. After 7 wk, milk yield of TRT and CON glands did not differ. Analysis of milk composition revealed that protein, lactose, fat and solids-not-fat percentages remained the same in TRT and CON glands. These results suggest that XS increases expression of milk synthesis genes, mammary stem/progenitor cells and has a small effect on milk yield.
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Expresión Génica/efectos de los fármacos , Cabras , Lactancia/genética , Glándulas Mamarias Animales/metabolismo , Ribonucleósidos/farmacología , Animales , Biomarcadores/análisis , Diferenciación Celular/genética , Proliferación Celular/genética , Femenino , Lactancia/efectos de los fármacos , Lactancia/fisiología , Glándulas Mamarias Animales/citología , Leche/química , Proteínas de la Leche/análisis , Proteínas de la Leche/genética , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Células Madre/fisiología , XantinasRESUMEN
Ergot alkaloids in endophyte-infected grasses inhibit prolactin secretion and reduce milk production in lactating cows. However, we previously showed that prepartum consumption of infected seed throughout the dry period did not inhibit subsequent milk production and prior exposure to bromocriptine (ergot peptide) actually increased production in the next lactation. To identify changes in the transcriptome and molecular pathways mediating the mammary gland's response to ergot alkaloids in the diet, RNA sequencing (RNA-seq) was performed on mammary tissues obtained from 22 multiparous Holstein cows exposed to 1 of 3 treatments. Starting at 90 ± 4 d prepartum, cows were fed endophyte-free fescue seed (control; CON), endophyte-free fescue seed plus 3×/wk subcutaneous injections of bromocriptine (BROMO; 0.1 mg/kg of BW), or endophyte-infected fescue seed (INF) as 10% of the diet. Cows were dried off 60 ± 2 d prepartum. Mammary biopsies from 4 (BROMO, INF) or 5 (CON) cows/treatment at each of the 3 phases were obtained: 7 d before dry off during the initial lactation (L1), mid-dry period (D), and 10 d postpartum (L2). Although tissue from the same cow was preferentially used at 3 phases (L1, D, L2), tissue from additional cows were used to as necessary to provide RNA of sufficient quality. Individual samples were used to generate individual RNA-seq libraries. Normalized reads of the RNA-seq data were organized into technical and biological replicates before processing with the RSEM software package. Each lactation phase was processed separately and genes that differed between any of 3 treatments were identified. A large proportion of genes differentially expressed in at least 1 treatment (n = 866) were found to be similarly expressed in BROMO and INF treatments, but differentially expressed from CON (n = 575, total for 3 phases). Of genes differentially expressed compared with CON, 104 genes were common to the L1 and L2 phases. Consistent with the production findings, networks most affected by treatments in L1 and L2 included lipid metabolism, small molecule biochemistry, and molecular transport, whereas networks related more to developmental and cellular functions and maintenance were evident during D phase. Similar patterns of expression in BROMO and INF during late and early lactation suggest involvement of similar cell signaling pathways or mechanisms of action for BROMO and INF and the importance of prolactin messaging pathways.
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Bovinos/fisiología , Endófitos/fisiología , Festuca/fisiología , Leche/metabolismo , Animales , Bovinos/genética , Bovinos/microbiología , Dieta/veterinaria , Femenino , Lactancia , Periodo Posparto , Semillas/microbiología , Análisis de Secuencia de ARN/veterinariaRESUMEN
BACKGROUND: Xanthosine treatment has been previously reported to increase mammary stem cell population and milk production in cattle and goats. However, the underlying molecular mechanisms associated with the increase in stem cell population and milk production remain unclear. METHODS: Primiparous Beetal goats were assigned to the study. Five days post-partum, one mammary gland of each goat was infused with xanthosine (TRT) twice daily (2×) for 3 days consecutively, and the other gland served as a control (CON). Milk samples from the TRT and CON glands were collected on the 10th day after the last xanthosine infusion and the total RNA was isolated from milk fat globules (MEGs). Total RNA in MFGs was mainly derived from the milk epithelial cells (MECs) as evidenced by expression of milk synthesis genes. Significant differentially expressed genes (DEGs) were subjected to Gene Ontology (GO) terms using PANTHER and gene networks were generated using STRING db. RESULTS: Preliminary analysis indicated that each individual goat responded to xanthosine treatment differently, with this trend being correlated with specific DEGs within the same animal's mammary gland. Several pathways are impacted by these DEGs, including cell communication, cell proliferation and anti-microbials. CONCLUSIONS: This study provides valuable insights into transcriptomic changes in milk producing epithelial cells in response to xanthosine treatment. Further characterization of DEGs identified in this study is likely to delineate the molecular mechanisms of increased milk production and stem or progenitor cell population by the xanthosine treatment.
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Xanthosine is hypothesized to increase stem cell number by promoting symmetrical cell division. Stem cells, in particular mammary stem/progenitor cells are important for gland growth and tissue repair. Molecular mechanism of xanthosine effects on mammary tissue is very limited therefore, a detailed study is warranted. The objective of this study was to evaluate transcriptomic changes in mammary gland infused/not infused with xanthosine of lactating goat. Seven primiparous Beetal goats on day 5 after kidding, were selected for the study. One gland of each goat was infused with xanthosine (TRT gland) twice daily for 3 days while the other gland did not receive any xanthosine and served as control (CON gland). Biopsy of mammary tissues was taken from TRT and CON glands, 2 days after the last day of treatment that is on day 10 after kidding. Illumina RNA-sequencing (RNA-seq) was performed for global gene expression analysis of contralateral glands. Of 382 differentially expressed genes (DEGs), 372 genes were annotated to the goat genome. Gene ontology analyses revealed majority of the DEGs to be associated with metabolic pathways (glycan and lipid metabolism), biosynthesis of antibiotics and peroxisome proliferator-activated receptor signalling pathways. These molecular pathways are either directly or indirectly involved with lipid metabolism in mammary tissue and host adaptive immune response. Expression of stem cell marker namely aldehyde dehydrogenase enzymes (ALDH1A1, ALDH3B1) were upregulated in the treatment gland. Real-time quantitative PCR (RT-qPCR) analyses of selected DEGs showed their expression profiles to be in agreement with results of RNA-seq. To our knowledge, this is the first study that describes effects of xanthosine on transcriptomic changes of mammary tissue. This information can be used further to dissect the molecular mechanisms underlying effects of xanthosine to improve production potential and udder health.
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Cabras/metabolismo , Lactancia/genética , Ribonucleósidos/farmacología , Animales , Femenino , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/genética , Ontología de Genes , Genoma , Cabras/genética , Lactancia/efectos de los fármacos , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/fisiología , Redes y Vías Metabólicas , Reacción en Cadena en Tiempo Real de la Polimerasa , Ribonucleósidos/metabolismo , Análisis de Secuencia de ARN , Transducción de Señal , Células Madre/citología , Transcriptoma/efectos de los fármacos , Transcriptoma/genética , XantinasRESUMEN
Ergot alkaloids in endophyte-infected grasses inhibit prolactin (PRL) secretion and may reduce milk production of cows consuming these grasses. We investigated the effects of consuming endophyte-infected fescue seed during late lactation and the dry period on mammary growth, differentiation, and milk production. Twenty-four multiparous Holstein cows were randomly assigned to 3 treatment groups. Starting at 90±4 d prepartum, cows were fed endophyte-free fescue seed (control; CON), endophyte-free fescue seed plus 3×/wk subcutaneous injections of bromocriptine (0.1mg/kg of body weight, positive control; BROMO), or endophyte-infected fescue seed (INF) as 10% of the diet on an as fed basis. Although milk yield of groups did not differ before treatment, at dry off (-60 d prepartum) INF and BROMO cows produced less milk than CON. Throughout the treatment period, basal concentrations of PRL and the prepartum increase in plasma PRL were reduced in INF and BROMO cows compared with CON cows. Three weeks after the end of treatment, circulating concentrations of PRL were equivalent across groups. In the subsequent lactation milk yield was not decreased; in fact, BROMO cows exhibited a 9% increase in milk yield relative to CON. Evaluation of mammary tissue during the dry period and the subsequent lactation, by quantitative histology and immunohistochemical analysis of proliferation markers and putative mammary stem or progenitor cell markers, indicated that feeding endophyte-infected fescue seed did not significantly affect mammary growth and development. Feeding endophyte-infected grasses during the dry period may permit effective utilization of feed resources without compromising milk production in the next lactation.
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Bovinos/fisiología , Endófitos/fisiología , Festuca/microbiología , Lactancia/efectos de los fármacos , Glándulas Mamarias Animales/efectos de los fármacos , Semillas/microbiología , Alimentación Animal/análisis , Animales , Bovinos/crecimiento & desarrollo , Dieta/veterinaria , Femenino , Glándulas Mamarias Animales/crecimiento & desarrollo , Distribución AleatoriaRESUMEN
Buffaloes account for more than 56% of total milk production in India. Cyclic remodeling of mammary glands of human, mice, cow, sheep, and goat is determined by mammary stem cells. It is logical to assume that buffalo mammary gland will have mammary stem/progenitor cells. Thus far, no report exists on identification of buffalo mammary stem cells. Hepatocyte nuclear factor 4 alpha (HNF4A) is a candidate marker for hepatic progenitor cells and has recently been suggested as a marker of bovine mammary stem/progenitor cells. We hypothesized that ( 1 ) HNF4A identifies putative buffalo mammary stem/progenitor cells and ( 2 ) the number of HNF4A-positive cells increases during mastitis. Sixteen buffalo mammary samples were collected from a local slaughterhouse. Hematoxylin and eosin staining were performed on 5-micron thick sections and on the basis of gross examination and histomorphology of the mammary glands, physiological stages of the animals were estimated as non-lactating (n = 4), mastitis (n = 9), and prepubertal (n = 3). In total, 24048 cells were counted (5-10 microscopic fields/animal; n = 16 animals) of which, 40% cells were mammary epithelial cells (MEC) and 60% cells were the stromal cells. The percentage of MEC in non-lactating animals was higher compared to mastitic animals (47.3% vs. 37.3%), which was likely due to loss of MEC in mastitis. HNF4A staining was observed in nuclei of MEC of ducts, alveoli, and stromal cells. Basal location and low frequency of HNF4A-positive MEC (ranges from 0.4-4.5%) were consistent with stem cell characteristics. Preliminary study showed coexpression of HNF4A with MSI1 (a mammary stem cell marker in sheep), suggesting HNF4A was likely to be a putative mammary stem/progenitor cell marker in buffalo. HNF4A-positive MEC (basal and luminal; light and dark stained) tended to be higher in non-lactating than the mastitic animals (8.73 ± 1.71% vs. 4.29 ± 1.19%; P = 0.07). The first hypothesis that HNF4A identify putative mammary stem/progenitor cells was confirmed but the second hypothesis that the number of mammary stem/progenitor cells decreases during mastitis was unsupported. This is the first report outlining the expression of HNF4A and identification of putative mammary stem/progenitor cells in buffalo mammary gland.
Asunto(s)
Biomarcadores/metabolismo , Búfalos/metabolismo , Factor Nuclear 4 del Hepatocito/metabolismo , Glándulas Mamarias Animales/metabolismo , Células Madre/metabolismo , Animales , Biomarcadores/análisis , Femenino , Factor Nuclear 4 del Hepatocito/análisis , Glándulas Mamarias Animales/química , Glándulas Mamarias Animales/citología , Células Madre/citologíaRESUMEN
Identification and characterization of mammary stem cells and progenitor cells from dairy animals is important in the understanding of mammogenesis, tissue turnover, lactation persistency and regenerative therapy. It has been realized by many investigators that altered lactation, long dry periods (non-milking period between two consecutive lactation cycles), abrupt cessation of lactation (common in water buffaloes) and disease conditions like mastitis, greatly reduce milk yield thus render huge financial losses within the dairy sector. Cellular manipulation of specialized cell types within the mammary gland, called mammary stem cells (MaSCs)/progenitor cells, might provide potential solutions to these problems and may improve milk production. In addition, MaSCs/progenitor cells could be used in regenerative therapy against tissue damage caused by mastitis. This review discusses methods of MaSC/progenitor cell manipulation and their mechanisms in bovine and caprine animals. Author believes that intervention of MaSCs/progenitor cells could lessen the huge financial losses to the dairy industry globally.
RESUMEN
BACKGROUND: Previous molecular characterizations of mammary stem cells (MaSC) have utilized fluorescence-activated cell sorting or in vitro cultivation of cells from enzymatically dissociated tissue to enrich for MaSC. These approaches result in the loss of all histological information pertaining to the in vivo locale of MaSC and progenitor cells. Instead, we used laser microdissection to excise putative progenitor cells and control cells from their in situ locations in cryosections and characterized the molecular properties of these cells. MaSC/progenitor cells were identified based on their ability to retain bromodeoxyuridine for an extended period. RESULTS: We isolated four categories of cells from mammary epithelium of female calves: bromodeoxyuridine label retaining epithelial cells (LREC) from basal (LRECb) and embedded layers (LRECe), and epithelial control cells from basal and embedded layers. Enriched expression of genes in LRECb was associated with stem cell attributes and identified WNT, TGF-ß, and MAPK pathways of self renewal and proliferation. Genes expressed in LRECe revealed retention of some stem-like properties along with up-regulation of differentiation factors. CONCLUSION: Our data suggest that LREC in the basal epithelial layer are enriched for MaSC, as these cells showed increased expression of genes that reflect stem cell attributes; whereas LREC in suprabasal epithelial layers are enriched for more committed progenitor cells, expressing some genes that are associated with stem cell attributes along with those indicative of cell differentiation. Our results support the use of DNA label retention to identify MaSC and also provide a molecular profile and novel candidate markers for these cells. Insights into the biology of stem cells will be gained by confirmation and characterization of candidate MaSC markers identified in this study.
RESUMEN
Nematode infections in ruminants are a major impediment to the profitable production of meat and dairy products, especially for small farms. Gastrointestinal parasitism not only negatively impacts weight gain and milk yield, but is also a major cause of mortality in small ruminants. The current parasite control strategy involves heavy use of anthelmintics that has resulted in the emergence of drug-resistant parasite strains. This, in addition to increasing consumer demand for animal products that are free of drug residues has stimulated development of alternative strategies, including selective breeding of parasite resistant ruminants. The development of protective immunity and manifestations of resistance to nematode infections relies upon the precise expression of the host genome that is often confounded by mechanisms simultaneously required to control multiple nematode species as well as ecto- and protozoan parasites, and microbial and viral pathogens. Understanding the molecular mechanisms underlying these processes represents a key step toward development of effective new parasite control strategies. Recent progress in characterizing the transcriptome of both hosts and parasites, utilizing high-throughput microarrays and RNA-seq technology, has led to the recognition of unique interactions and the identification of genes and biological pathways involved in the response to parasitism. Innovative use of the knowledge gained by these technologies should provide a basis for enhancing innate immunity while limiting the polarization of acquired immunity can negatively affect optimal responses to co-infection. Strategies for parasite control that use diet and vaccine/adjuvant combination could be evaluated by monitoring the host transcriptome for induction of appropriate mechanisms for imparting parasite resistance. Knowledge of different mechanisms of host immunity and the critical regulation of parasite development, physiology, and virulence can also selectively identify targets for parasite control. Comparative transcriptome analysis, in concert with genome-wide association (GWS) studies to identify quantitative trait loci (QTLs) affecting host resistance, represents a promising molecular technology to evaluate integrated control strategies that involve breed and environmental factors that contribute to parasite resistance and improved performance. Tailoring these factors to control parasitism without severely affecting production qualities, management efficiencies, and responses to pathogenic co-infection will remain a challenge. This review summarizes recent progress and limitations of understanding regulatory genetic networks and biological pathways that affect host resistance and susceptibility to nematode infection in ruminants.