The Improved Milk Quality and Enhanced Anti-Inflammatory Effect in Acetylserotonin-O-methyltransferase (ASMT) Overexpressed Goats: An Association with the Elevated Endogenous Melatonin Production.

BACKGROUND: Transgenic animal production is an important means of livestock breeding and can be used to model pharmaceutical applications. METHODS: In this study, to explore the biological activity of endogenously produced melatonin, Acetylserotonin-O-methyltransferase (ASMT)-overexpressed melatonin-enriched dairy goats were successfully generated through the use of pBC1-ASMT expression vector construction and prokaryotic embryo microinjection. RESULTS: These transgenic goats have the same normal phenotype as the wild-type goats (WT). However, the melatonin levels in their blood and milk were significantly increased (p < 0.05). In addition, the quality of their milk was also improved, showing elevated protein content and a reduced somatic cell number compared to the WT goats. No significant changes were detected in the intestinal microbiota patterns between groups. When the animals were challenged by the intravenous injection of E. coli, the ASMT-overexpressed goats had a lower level of pro-inflammatory cytokines and higher anti-inflammatory cytokines compared to the WT goats. Metabolic analysis uncovered a unique arachidonic acid metabolism pattern in transgenic goats. CONCLUSIONS: The increased melatonin production due to ASMT overexpression in the transgenic goats may have contributed to their improved milk quality and enhanced the anti-inflammatory ability compared to the WT goats.

Responses of Transgenic Melatonin-Enriched Goats on LPS Stimulation and the Proteogenomic Profiles of Their PBMCs.

The anti-inflammatory activity of melatonin (MT) has been well documented; however, little is known regarding endogenously occurring MT in this respect, especially for large animals. In the current study, we created a MT-enriched animal model (goats) overexpressing the MT synthetase gene Aanat. The responses of these animals to lipopolysaccharide (LPS) stimulation were systematically studied. It was found that LPS treatment exacerbated the inflammatory response in wild-type (WT) goats and increased their temperature to 40 °C. In addition, their granulocyte counts were also significantly elevated. In contrast, these symptoms were not observed in transgenic goats with LPS treatment. The rescue study with MT injection into WT goats who were treated with LPS confirmed that the protective effects in transgenic goats against LPS were attributed to a high level of endogenously produced MT. The proteomic analysis in the peripheral blood mononuclear cells (PBMCs) isolated from the transgenic animals uncovered several potential mechanisms. MT suppressed the lysosome formation as well as its function by downregulation of the lysosome-associated genes Lysosome-associated membrane protein 2 (LAMP2), Insulin-like growth factor 2 receptor (IGF2R), and Arylsulfatase B (ARSB). A high level of MT enhanced the antioxidant capacity of these cells to reduce the cell apoptosis induced by the LPS. In addition, the results also uncovered previously unknown information that showed that MT may have protective effects on some human diseases, including tuberculosis, bladder cancer, and rheumatoid arthritis, by downregulation of these disease-associated genes. All these observations warranted further investigations.

Whole-genome methylation analysis reveals epigenetic variation between wild-type and nontransgenic cloned, ASMT transgenic cloned dairy goats generated by the somatic cell nuclear transfer.

BACKGROUND: SCNT (somatic cell nuclear transfer) is of great significance to biological research and also to the livestock breeding. However, the survival rate of the SCNT cloned animals is relatively low compared to other transgenic methods. This indicates the potential epigenetic variations between them. DNA methylation is a key marker of mammalian epigenetics and its alterations will lead to phenotypic differences. In this study, ASMT (acetylserotonin-O-methyltransferase) ovarian overexpression transgenic goat was produced by using SCNT. To investigate whether there are epigenetic differences between cloned and WT (wild type) goats, WGBS (whole-genome bisulfite sequencing) was used to measure the whole-genome methylation of these animals. RESULTS: It is observed that the different mCpG sites are mainly present in the intergenic and intronic regions between cloned and WT animals, and their CG-type methylation sites are strongly correlated. DMR (differentially methylated region) lengths are located around 1000 bp, mainly distributed in the exonic, intergenic and intronic functional domains. A total of 56 and 36 DMGs (differentially methylated genes) were identified by GO and KEGG databases, respectively. Functional annotation showed that DMGs were enriched in biological-process, cellular-component, molecular-function and other signaling pathways. A total of 10 identical genes related to growth and development were identified in GO and KEGG databases. CONCLUSION: The differences in methylation genes among the tested animals have been identified. A total of 10 DMGs associated with growth and development were identified between cloned and WT animals. The results indicate that the differential patterns of DNA methylation between the cloned and WT goats are probably caused by the SCNT. These novel observations will help us to further identify the unveiled mechanisms of somatic cell cloning technology, particularly in goats.