The inferior oocytes (IOs), which are not suitable for embryo development, occupy roughly one-third or more of the collected immature bovine oocytes. The IOs are usually discarded from the in vitro bovine embryo production process. Improving the quality of the inferior oocytes (IOs) and make them available in in vitro embryo production would have important biological, as well as commercial, value. This study was designed to investigate whether melatonin could improve the quality of IOs and make them usable in the in vitro maturation (IVM) and subsequent (in vitro fertilization) IVF embryo development. The results indicated that: the maturation rate of IOs and their subsequent IVF embryo developments were impaired compared to cumulus-oocyte complexes and melatonin treatment significantly improved the quality of IOs, as well as their IVF and embryo developments. The potential mechanisms are that: (1) melatonin reduced reactive oxygen species (ROS) and enhanced glutathione (GSH) levels in the IOs, thereby protecting them from oxidative stress; (2) melatonin improved mitochondrial normal distribution and function to increase ATP level in IOs; and (3) melatonin upregulated the expression of ATPase 6, BMP-15, GDF-9, SOD-1, Gpx-4, and Bcl-2, which are critical genes for oocyte maturation and embryo development and downregulated apoptotic gene expression of caspase-3.

The in vitro maturation efficiency of porcine oocytes is relatively low, and this limits the production of in vitro porcine embryos. Since melatonin is involved in mammalian reproductive physiology, in this study, we have explored whether endogenously produced melatonin can help in porcine oocyte in vitro maturation. We have found, for the first time in the literature, that mitochondria are the major sites for melatonin biosynthesis in porcine oocytes. This mitochondrially originated melatonin reduces ROS production and increases the activity of the mitochondrial respiratory electron transport chain, mitochondrial biogenesis, mitochondrial membrane potential, and ATP production. Therefore, melatonin improves the quality of oocytes and their in vitro maturation. In contrast, the reduced melatonin level caused by siRNA to knockdown AANAT (siAANAT) is associated with the abnormal distribution of mitochondria, decreasing the ATP level of porcine oocytes and inhibiting their in vitro maturation. These abnormalities can be rescued by melatonin supplementation. In addition, we found that siAANAT switches the mitochondrial oxidative phosphorylation to glycolysis, a Warburg effect. This metabolic alteration can also be corrected by melatonin supplementation. All these activities of melatonin appear to be mediated by its membrane receptors since the non-selective melatonin receptor antagonist Luzindole can blunt the effects of melatonin. Taken together, the mitochondria of porcine oocytes can synthesize melatonin and improve the quality of oocyte maturation. These results provide an insight from a novel aspect to study oocyte maturation under in vitro conditions.

In this study, the effects of daily melatonin supplementation (2 mg/kg) at the late gestational stage on the reproductive performance of the sows have been investigated. This treatment potentially increased the litter size and birth survival rate and significantly increased the birth weight as well as the weaning weight and survival rate of piglets compared to the controls. The mechanistic studies have found that these beneficial effects of melatonin are not mediated by the alterations of reproductive hormones of estrogen and progesterone, nor did the glucose and lipid metabolisms, but they were the results of the reduced oxidative stress in placenta associated with melatonin supplementation. Indeed, the melatonergic system, including mRNAs and proteins of AANAT, MTNR1A and MTNR1B, has been identified in the placenta of the sows. The RNA sequencing of placental tissue and KEGG analysis showed that melatonin activated the placental tissue fluid shear stress pathway to stimulate the Nrf2 signaling pathway, which upregulated its several downstream antioxidant genes, including MGST1, GSTM3 and GSTA4, therefore, suppressing the placental oxidative stress. All these actions may be mediated by the melatonin receptor of MTNR1B.

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.

Melatonin is synthesized in multiple tissues and organs of pigs, and existing studies have shown the presence of the melatonin-synthesizing enzyme ASMT protein. However, the genomic information for the ASMT gene has been lacking. The aim of this study was to locate the genomic information of the ASMT gene in pigs using comparative genomics analysis and then obtain the coding region information through molecular cloning. First, using the NCBI Genome Data Viewer, we found that in most animals, the AKAP17A gene is often located next to the ASMT gene, with both genes arranged in the same direction. Similarly, the P2RY8 gene is commonly adjacent to the ASMTL gene, also in the same orientation. We also discovered that the ASMTL gene is frequently adjacent to the ASMT gene and arranged in the opposite direction. Using the “three-point localization” principle, we inferred the position of the ASMT gene based on the coordinates of AKAP17A and ASMTL in pigs. Our results revealed that on the pig X chromosome, a gene called LOC110258194 is located next to the AKAP17A and ASMTL genes, and its arrangement aligns with the ASMT gene in other species. Additionally, Ensembl contains a gene, ENSSSCG00000032659, at the same position, with completely overlapping exons, though it is not annotated as ASMT. Further analysis using the TreeFam tool from EMBL-EBI and the CDD tool from NCBI revealed that LOC110258194 and ENSSSCG00000032659 do not contain the typical Maf domain of ASMTL and, thus, should not be annotated as ASMTL, but rather as the ASMT gene. Using a slow-down PCR method for high-GC content genes, we successfully cloned the full CDS region of the pig ASMT gene and identified a new transcript missing Exon 6 and Exon 7. This transcript was submitted to NCBI and assigned the GenBank accession number MW847601. Our results represent the first successful localization of the ASMT gene in pigs, the first cloning of the ASMT gene’s coding region, and the first discovery of a new transcript of the pig ASMT gene.