In Vivo and In Vitro Expression of iC1, a Methylation-Controlled J Protein (MCJ) in Bovine Liver, and Response to In Vitro Bovine Fatty Liver Disease Model.

Mitochondrial complex I inhibitor (iC1) is a methylation-controlled J protein (MCJ) that decreases cellular respiration by inhibiting oxidative phosphorylation. Recent rodent studies showed that loss or inhibition of iC1 was associated with preventing lipid accumulation. A common metabolic disorder of dairy cattle is a fatty liver disease (FLD), which often occurs during the periparturient period. In humans and rodents, iC1 is expressed in the liver and acts as a mitochondrial "brake". However, iC1 expression in bovine liver and its possible role in FLD development have not yet been characterized. We hypothesized that iC1 is expressed in the bovine liver and that the expression of iC1 is correlated with FLD in periparturient dairy cattle. To test this hypothesis, we collected bovine liver tissue samples from an abattoir and isolated primary hepatic cells immediately following harvest. Utilizing an in vitro model of bovine FLD developed in our laboratory, we cultured primary hepatic cells in low-glucose DMEM supplemented with 10% FBS. The basal media was made to induce lipid accumulation and cytotoxicity in the primary liver cells with three treatments. To the basal media (control) we added 0.4 mM palmitate (treatment 1) or 20 ng/mL TNFα (treatment 2), or both 0.4 mM palmitate and 20 ng/mL TNFα (treatment 3). Consistent with our hypothesis, we present the novel characterization of iC1 expression in primary bovine liver cells cultured with or without the addition of lipotoxic factors made to emulate bovine FLD. We demonstrate both in situ and in vitro expression of iC1 in bovine liver and mRNA expression in hepatic cells and in the precipitates of conditioned media. The results of RT-qPCR, IHC, and western blot all demonstrated the expression of iC1 in bovine liver. In addition, we isolated precipitates of conditioned media further demonstrated iC1 expression by RT-qPCR. The transcript of iC1 tended to be more concentrated (4-fold; p > 0.05) in TNFα-treated conditioned media when compared with the control. Taken together, we present the novel finding that iC1 transcript and protein are expressed in liver tissue from dairy cattle, primary hepatic cells isolated from that liver tissue, and, finally, in the conditioned media derived from those cells. These novel findings and the prior findings on the role of iC1 in rodents and humans indicate that further investigation of the role of iC1 in the etiology and pathology of FLD in periparturient dairy cows is warranted.

Understanding, Status, and Therapeutic Potentials of Stem Cells in Goat.

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.

Significance and Relevance of Spermatozoal RNAs to Male Fertility in Livestock.

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.

High expression of aldehyde dehydrogenase 1 and tissue necrosis factor alpha may relate to chronic infection of buffalo mammary gland.

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.

Deciphering the transcriptome of prepubertal buffalo mammary glands using RNA sequencing.

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.

Evaluation of xanthosine treatment on gene expression of mammary glands in early lactating goats.

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.

Examination of the xanthosine response on gene expression of mammary epithelial cells using RNA-seq technology.

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.

In vivo response of xanthosine on mammary gene expression of lactating Beetal goat.

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.

Imidacloprid induced histomorphological changes and expression of TLR-4 and TNFα in lung.

The imidacloprid is used worldwide as a pesticide and has been linked with endocrine disturbances and reduced pulmonary function. However, effects of imidacloprid alone or in combination with microbial molecules on lungs are not fully understood. Because the pulmonary effects of interactions of endotoxins with imidacloprid are unknown, we designed a study to investigate that in a mouse model. Mice (N=14) were given imidacloprid orally @ 1/20(th) of LD50 dissolved in corn oil for 30days. After the treatments, six animals from each group were challenged with E. coli lipopolysaccharide (LPS) @ 80µg/animal via intranasal route and remaining animals were challenged with normal saline solution @ 80µl/animal via same route. Imidacloprid in combination with LPS led to significant increase in total cell and neutrophil counts in BAL and peripheral blood. Semi-quantitative histopathology revealed lung injury in imidacloprid treatment group and injury was more marked in animal receiving both imidacloprid and LPS. There was no change (p<0.05) in the expression of TLR-4 and TNF-α both at mRNA and protein levels following exposure to imidacloprid alone or in combination with LPS. The data show that imidacloprid alone or in combination with LPS resulted changes in lung morphology without altering the expression of TLR-4 and TNF-α. Furthermore, pre-treatment with imidacloprid didn't affect response to LPS.

Expression of Putative Stem Cell Marker, Hepatocyte Nuclear Factor 4 Alpha, in Mammary Gland of Water Buffalo.

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.

Isolation and RFLP genotyping of Toxoplasma gondii from the mongoose (Herpestes auropunctatus) in Grenada, West Indies.

Little is known of the genetic diversity and epidemiology of Toxoplasma gondii infection in wildlife in Caribbean Islands. The prevalence and genetic diversity of T. gondii in mongooses (Herpestes auropunctatus) was investigated. During 2011 and 2012, 91 mongooses were trapped in different parts of Grenada, bled, euthanized, and examined at necropsy. Antibodies to T. gondii were found in 27 mongooses tested by the modified agglutination test (cut-off titer 25). Muscles (heart, tongue, neck) of 25 of the seropositive mongooses were bioassayed for T. gondii infection in mice. Viable T. gondii was isolated by bioassay in mice from four mongooses with MAT titers of 1:50 in two, 1:200 for one, and 1:400 for one mongoose. The four T. gondii isolates were further propagated in cell culture. Strain typing of T. gondii DNA extracted from cell-cultured tachyzoites using the 10 PCR-restriction fragment length polymorphism (RFLP) markers SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico revealed one isolate belongs to the Type III (ToxoDB #2) lineage, two to ToxoDB#7 lineage, and one to the ToxoDB #216 lineage. This is the first report of T. gondii isolation and genotyping in H. auropunctatus worldwide.