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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.

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.

Each quarter of the bovine mammary gland is an anatomically and functionally distinct gland. However, mastitis in one quarter may affect function of adjacent, uninfected glands. To investigate the mechanisms and potential mediators of these effects, we quantified early responses of the mammary gland to intramammary lipopolysaccharide (LPS) challenge, distinguishing between local and systemic effects. Ten multiparous cows over 70 d in milk were blocked into pairs by breed, cow-level somatic cell count (SCC), and milk yield. Within block, one cow was assigned to LPS treatment (T) such that both the front and the rear quarter of a randomly selected udder half received an infusion of 50 µg of LPS in 10 mL of saline (T-L); the contralateral quarters received only 10 mL of saline (T-S). Similarly, each paired control cow (C) received either 10 mL of saline (C-S) or no infusion (C-N) into udder halves. Cows were quarter milked twice daily, with foremilk samples (∼30 mL, front quarters) taken at -24, 0, 3, 6, 12, and 24 h relative to infusions. At 24 h, average milk yield in T-L and T-S quarters fell to 23 and 32% of pre-infusion levels, respectively. For T cows, systemic effects were observed by 3 h post-infusion as rectal temperature was elevated and foremilk fat concentration was reduced in both T-L and T-S. However, SCC and concentrations of l-lactate and total protein in foremilk indicated a local response to LPS: protein was transiently higher at 3 h, whereas SCC and lactate were higher at 6 h in T-L compared with T-S. Lactose concentration showed a local effect at 6 h, being lower in T-L than in T-S, and then a systemic effect at 12 h, being lower in both T-L and T-S than C quarters. Concomitant with changes in milk, systemic effects were also observed in blood. Plasma antioxidant potential and glucose concentration were lower in T cows than in C cows at 6 or 12 h, respectively, although neither variable remained different at 24 h. In summary, unilateral LPS infusion induced distinct, time-dependent effects on each milk component. Depending on the component, effects were local, systemic, or both, suggesting involvement of multiple different mediators that collectively result in systemic inhibition of milk production.

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 (

Abstract: 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.

Lysyl oxidase (LOX) is an extracellular metalloenzyme which mediates crosslinking of collagen and elastin. It has been reported to play a pivotal role in cancer metastasis especially in women suffering from breast cancer. The present study is the first to evaluate the gene expression levels of LOX by Real time-polymerase chain reaction (Real time-PCR) in dogs with mammary tumor besides molecular cloning and expression of canine lysyl oxidase gene (lox). Real time-PCR studies showed a significant upregulation (threefold higher) of lox in mammary tumor cases as compared to healthy dogs indicating its possible diagnostic and prognostic role in canine mammary tumors (CMTs). Cloning and sequencing of lox gene revealed 1230 bp CDS which is mostly conserved in C-terminal region. Sequence analysis of canine lox showed that it shares 99% homology with the predicted sequence available on NCBI and had greatest identity with the lox gene from cat. Protein structure predicted with homology modelling was validated by Ramachandran plot analysis which revealed most (approximately 95%) of the amino acids in favoured region. Additionally, recombinant lysyl oxidase expressed as His-tagged fusion protein in prokaryotic expression vector (pPROExHTa) was used in an ELISA for detection of circulating protein LOX in serum of CMT subjects. Receiver operating characteristics analysis of the ELISA revealed high sensitivity (90%) and specificity (85%) with histopathology as reference standard. Taken together, we propose LOX as a diagnostic biomarker and a putative prognostic candidate in CMT cases.

Infusion of lipopolysaccharides (LPS) into a mammary gland can provoke inflammatory responses and impair lactation in both the infused gland and neighboring glands. To gain insight into the mechanisms controlling the spatiotemporal response to localized mastitis in lactating dairy cows, we performed RNA sequencing on mammary tissue from quarters infused with LPS, neighboring quarters in the same animals, and control quarters from untreated animals at 3 and 12 h postinfusion. Differences in gene expression were annotated to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Comparing mammary transcriptomes from all 3 treatments revealed 3,088 and 1,644 differentially expressed (DE) genes at 3 and 12 h, respectively. Of these genes, >95% were DE only in LPS-infused quarters and represented classical responses to LPS: inflammation, apoptosis, tissue remodeling, and altered cell signaling and metabolism. Although relatively few genes were DE in neighboring quarters (56 at 3 h; 74 at 12 h), these represented several common pathways. At 3 h, tumor necrosis factor (TNF), nuclear factor-κB, and nucleotide-binding and oligomerization domain (NOD)-like receptor signaling pathways were identified by the upregulation of anti-inflammatory (NFKBIA, TNFAIP3) and cell adhesion molecule (VCAM1, ICAM1) genes in neighboring glands. Additionally, at 12 h, several genes linked to 1-carbon and serine metabolism were upregulated. Some responses were also regulated over time. The proinflammatory response in LPS-infused glands diminished between 3 and 12 h, indicating tight control over transcription to re-establish homeostasis. In contrast, 2 glucocorticoid-responsive genes, FKBP5 and ZBTB16, were among the top DE genes upregulated in neighboring quarters at both time points, indicating potential regulation by glucocorticoids. We conclude that a transient, systemic immune response was sufficient to disrupt lactation in neighboring glands. This response may be mediated directly by proinflammatory factors from the LPS-infused gland or indirectly by secondary factors released in response to systemic inflammatory signals.

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.

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.

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.

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.

Isolation of good quality ribonucleic acid (RNA) from mammary glands of elite animals is often hindered by invasive method of mammary tissue sampling, ethical permission, time consuming, and repeated biopsies from the same animal at different time points. The aim of the present study was to optimize a protocol for RNA isolation from milk fat that is suitable for transcriptome analysis. In this study, authors isolated good quality RNA from milk fat of goats and buffalo milk by combining two methods namely Trizol and GenElute mammalian RNA isolation kit. The results showed good quality as well as high quantity of total RNA suitable for downstream applications like reverse transcriptase-quantitative polymerase chain reaction and next generation sequencing.

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.

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.

Water buffaloes are the principle source of milk in south Asia and Africa. Mammary gland repeatedly undergoes the cycles of growth and regeneration during pregnancy, lactation and involution. It is assumed that buffalo mammary gland has mammary stem and progenitor cells that regulate gland growth and regeneration. In the present study the authors analyzed percentage of cellular composition, proliferation status and putative mammary stem/progenitor cell population. Identification of putative buffalo mammary stem/progenitor cells was attempted using immunohistochemical staining with Musashi1 (MSI1), an adult stem cell marker and fibronectin type III domain containing 3B (FNDC3B), a mammary stem and cancer cell marker. Immunolocalization of MSI1 and FNDC3B showed nuclear and cytoplasmic staining of alveolar and ductal mammary epithelial cells (MEC) and a few stromal cells. The percentage of MSI1-positive MEC in non-lactating (3.31 ± 1.11 %), lactating (2.73 ± 0.78 %) and mastitic glands (3.30 ± 0.97 %) were equivalent, indicating that the proportion of putative stem/progenitor cell population did not differ during various physiological stages. Likewise, the percentage of FNDC3B-positive MEC in non-lactating (12.40 ± 3.22 %) tended to be higher than lactating (8.19 ± 2.71 %) and mastitic glands (4.88 ± 2.37 %). In some cases, expression of MSI1 and FNDC3B was exceptionally high with high proliferative indices (37.6 ± 2.4 %)-an indication of tumor cells. This is the first report on expression of MSI1 and FNDC3B in buffalo mammary gland. Identification of buffalo mammary stem cells using MSI1 and FNDC3B requires further studies and functional validation.

The ω-3 rich plant oil supplementation in animal diet is a reliable feeding strategy to enhance ω-3 fatty acids content in milk and meat. To investigate oil induced differential gene expressions of milk protein genes (LALBA, CSN2) and fat synthesis genes (CD36, XDH, ACACA, SCD, FABP3) in mammary epithelial cells. Twelve Beetal goats were fed control diet (CON) and linseed oil (LSO) or chia oil (CO) supplemented diets (at 1% dry matter basis) for sixty days. Total RNA was isolated from milk fat, a rich source of mammary RNA, before (d 0) and after (d 15 and d 34) of the feeding. Supplementation of diet with ω-3 rich oils and or stage of lactation showed no significant (P > 0.05) differences in milk RNA quality and quantity Real time quantitative PCR (RT-qPCR) analysis showed up-regulation of ACACA, FABP3 and CD36 genes at d 15 in LSO supplemented milk, while expression of SCD and XDH was upregulated at d 34 in CO, supplemented milk with respective to control. Oil supplementation increased abundance of CD36 at d 34 in both the treatment groups (LSO vs. CON; 5-fold, P 0.01 and CO vs. CON; 4.2-fold, P 0.05), indicating involvement of CD36 in lipid metabolism. Expression of milk protein genes was though numerically high at d 34 in LSO and CO group (vs. CON), did not differ (P> 0.05) among groups. There were statistically significant interactions observed among all the genes in present study with the stage of lactation. These results suggest that regulation of milk protein and lipogenic genes in goats get affected by feeding ω-3 rich plant oils. Difference in the expression of these lipogenic genes may be beneficial in altering fatty acids compositions of goat milk for human health benefits.

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. Electronic supplementary material The online version of this article (10.1186/s40781-018-0177-5) contains supplementary material, which is available to authorized users.

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.

India is the largest producer of buffalo milk in the world. Buffalo milk is rich in minerals like calcium and magnesium, containing 100 % more fat and less cholesterol than cow milk. Physio-chemical properties of dairy products depend upon the size and distribution of milk fat globules (MFG). Individual MFG from Murrah buffalo were analysed as a function of size and distribution and comparisons were made with MFG of crossbred (HF X Sahiwal) cows. Crescents associated with MFG could serve as a source of RNA of mammary epithelial cells and that would be a non-invasive method of epithelial cell RNA. Acridine orange staining of fresh milk was visualized by light microscopy under red and green channel filters. MFG and MFG with crescents were analysed using imageJ image analysis software. The percentage of large sized MFG (area >20 µm2) was greater and the percentage of small sized MFG (area

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.

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.

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.

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 were associated with stem cell attributes and identified WNT, TGF-β 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. Conclusions: 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.

In the present study, we used high-throughput sequencing, miRNA-seq, to discover and explore the expression profiles of known and novel miRNAs in TLR ligand-stimulated vis-à-vis non-stimulated (i.e. Control) peripheral blood mononuclear cells (PBMCs) isolated from blood of healthy Murrah buffaloes. Six small RNA (sRNA) libraries were multiplexed in Ion Torrent PI chip and sequenced on Ion Proton System. The reads obtained were aligned to the Bos taurus genome (UMD3.1 assembly), which is phylogenetically closest species to buffalo (Bubalus bubalis). A total of 160 bovine miRNAs were biocomputationally identified in buffalo PBMCs and 130 putatively novel miRNAs (not enlisted in the bovine mirBase) were identified. All of these 290 miRNAs identified across the six treatment and control samples represent the repertoire of novel miRNAs for the buffalo species. The expression profiles of these miRNAs across the samples have been represented by sample dendrogram and heatmap plots. The uniquely expressed miRNAs in each treatment and control groups were identified. A few miRNAs were expressed at very high levels while the majority of them were moderately expressed. The miRNAs bta-miR-103 and -191 were found to be highly abundant and expressed in all the samples. Other abundantly expressed miRNAs include bta-miR-19b, -29b, -15a, -19a, -30d, -30b-5p and members of let family (let 7a-5p, let 7g & let 7f) in LPS and CpG treated PBMCS and bta-miR-191, -103 & -19b in Poly I:C stimulated PBMCs. Only one novel miRNA (bta-miR-11039) out of 130 identified putatively novel miRNAs, was expressed in all the six samples and differentially expressed (>2- fold) miRNAs were identified. Six of the differentially expressed miRNAs across the groups (bta-miR-421, bta-let-7i, bta-miR-138, bta-miR-21-5p, bta-miR-222 and bta-miR-27b) were subsequently confirmed by TaqMan quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, the target genes of differentially expressed miRNAs were enriched for the roles in innate immunity and TLR signaling pathways. This maiden study on profiling and cataloguing of bubaline miRNAs expressed in TLR-ligand stimulated PBMCs will provide an important reference point for future studies on regulatory roles of miRNAs in immune system of buffaloes.

Mammary stem cells (MaSC) provide for net growth, renewal and turnover of mammary epithelial cells, and are therefore potential targets for strategies to increase production efficiency. Appropriate regulation of MaSC can potentially benefit milk yield, persistency, dry period management and tissue repair. Accordingly, we and others have attempted to characterize and alter the function of bovine MaSC. In this review, we provide an overview of current knowledge of MaSC gained from studies using mouse and human model systems and present research on bovine MaSC within that context. Recent data indicate that MaSC retain labeled DNA for extended periods because of their selective segregation of template DNA strands during mitosis. Relying on this long-term retention of bromodeoxyuridine-labeled DNA, we identified putative bovine MaSC. These label-retaining epithelial cells (LREC) are in low abundance within mammary epithelium (

Genomic structural variation is an important and abundant source of genetic and phenotypic variation. We report an initial analysis of copy number variations (CNVs) in cattle selected for resistance or susceptibility to intestinal nematodes. We performed three array comparative genomic hybridization (CGH) experiments to compare Angus cattle with extreme phenotypes for fecal egg count and serum pepsinogen level. We identified 20 CNVs in total, of which 12 were within known chromosomes harboring or adjacent to gains or losses. About 85% of the CNV identified (17/20) overlapped with cattle CNV regions that were reported recently. Selected CNVs were further validated by independent methods using quantitative PCR (qPCR) and FISH. Pathway analyses indicated that annotated cattle genes within these variable regions are particularly enriched for immune function affecting receptor activities, signal transduction, and transcription. Analysis of transcription factor binding sites (TFBS) within the promoter regions of differentially expressed genes suggested that common transcription factors are probably involved in parasite resistance. These results provide valuable hypotheses for the future study of cattle CNVs underlying economically important health and production traits.

A rapid method of 5-bromo-2'-deoxyuridine (BrdU) immunostaining was developed in cryosections of bovine mammary tissue while preserving RNA quality of the stained section. A thymidine analog that is incorporated into DNA of proliferating cells, BrdU serves as a proliferation marker. Immunostaining of BrdU-labeled cells within a histological section requires heat, enzymatic or chemical-mediated antigen retrieval to open double-stranded DNA, and exposure to the BrdU antigen. Although these established treatments permit staining, they preclude use of cells within the tissue section for further gene expression experiments. Additionally, long antibody incubations and washing steps lead to extensive RNA degradation and elution. A protocol was developed for immunolocalization of BrdU-labeled cells in cryosections of bovine mammary tissue, which does not require harsh DNA denaturation and preserved RNA integrity and quantity. This protocol used an initial acetone:polyethylene glycol 300 [9:1 (vol/vol)] fixation (2 min) followed by staining with methyl green (0.5% aqueous; 2 min) to stabilize macromolecules, antigen retrieval with deionized formamide (70% in nuclease-free phosphate buffered saline; 4 min incubation), antibody incubation in the presence of RNase inhibitors (5 min), and minimal washing to facilitate recovery of RNA from cells from the stained sections. Applicability of this protocol to other nuclear antigens was evaluated by testing its suitability for staining estrogen receptor alpha and Ki-67 antigen. In both cases, use of the protocol provided good immunostaining and tissue morphology. The RNA quality of estrogen receptor alpha- and Ki-67-stained sections was not evaluated. Quality of the isolated RNA from BrdU-stained sections was evaluated by micro-fluidic electrophoresis and its utility was confirmed using quantitative reverse transcription-PCR. Staining intensity obtained with this labeling protocol was similar to that obtained using conventional immunohistochemistry protocols. When coupled with laser microdissection and RNA or cDNA amplification, this immunostaining protocol provided a means for future transcriptome analysis of BrdU-labeled cells within a complex tissue.

Department of Chemistry, Patna University, Patna-800 005 L. N. Mithila University, Darbhanga-846 004 Manuscript received 18 July 1996, revised 25 November 1997, accepted 30 April 1998 Complexes of trithiocarbodiglycolic acid (LH2) with bivalent metal ions of the formulae [ML(H2O)2] (M = Coll, Nill, Mn or CdII}and [CuL(H2O)] have been prepared and characterised by magnetic moment, it and electronic spectral data.

Background Mammary stem cells are critical for growth and maintenance of the mammary gland and therefore are of considerable interest for improving productivity and efficiency of dairy animals. Xanthosine treatment has been demonstrated to promote expansion of putative mammary stem cells in vivo, and hepatic and hair follicle stem cells in vitro. In the latter, xanthosine promoted the symmetrical division of hepatic and hair follicle stem cells. The objective of this study was to determine if treating primary cultures of bovine mammary epithelial cells (MEC) with xanthosine increases the stem/progenitor cell population by promoting symmetrical division of mammary stem cells. Results In vitro treatment with xanthosine increased the population of MEC during the exponential phase of cell growth, reducing the doubling time from 86 h in control cultures to 60 h in xanthosine-treated cultures. The bromodeoxyuridine (BrdU) labeling index and the proportion of MEC in S-phase both were increased by xanthosine treatment, indicating that increased cell accretion was due to increased cell proliferation. Analysis of daughter-pairs indicated that xanthosine promoted a shift from asymmetric to symmetric cell division. Moreover, the 30 % increase in symmetric cell division was concomitant with an increase in the proportion of MEC that were positive for a putative stem cell marker (FNDC3B) and a trend toward increased telomerase activity. These results suggest that xanthosine treatment in vitro can increase cell proliferation, promote symmetric cell division and enhance stem/progenitor cell activity. Conclusions Xanthosine treatment increased the proliferation rate of bovine MEC in vitro. This was likely to be mediated by an increase in the proportion of stem/progenitor cells in the MEC population due to promotion of symmetrical stem cell division by xanthosine.