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 the current study, using Aanat and Mt2 KO mice, we observed that the preservation of the melatonergic system is essential for successful early pregnancy in mice. We identified that aralkylamine N-acetyltransferase (AANAT), melatonin receptor 1A (MT1), and melatonin receptor 1B (MT2) were all expressed in the uterus. Due to the relatively weak expression of MT1 compared to AANAT and MT2, this study focused on AANAT and MT2. Aanat and Mt2 KO significantly reduced the early implantation sites and the abnormal morphology of the endometrium of the uterus. Mechanistical analysis indicated that the melatonergic system is the key player in the induction of the normal nidatory estrogen (E2) response for endometrial receptivity and functions by activating the STAT signaling pathway. Its deficiency impaired the interactions between the endometrium, the placenta, and the embryo. The reduction in melatonin production caused by Aanat KO and the impairment of signal transduction caused by Mt2 KO reduced the uterine MMP-2 and MMP-9 activity, resulting in a hyperproliferative endometrial epithelium. In addition, melatonergic system deficiency also increased the local immunoinflammatory reaction with elevated local proinflammatory cytokines leading to early abortion in the Mt2 KO mice compared to the WT mice. We believe that the novel data obtained from the mice might apply to other animals including humans. Further investigation into the interaction between the melatonergic system and reproductive effects in different species would be worthwhile.

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.

The negative energy balance occurring in the periparturient period of cows will impede their health and postpartum performance. To target this issue, L-tryptophan was supplied to the prepartum cows. The results showed that L-tryptophan supplementation significantly increased the serum melatonin level and was accompanied with increases in SOD activity, IL-10 and colostrum IgA levels as well as decreases in MDA and IL-6 levels compared to the control cows. The incidence of postpartum diseases was significantly lower and the pregnancy rate was significantly higher in cows fed L-tryptophan than in the control group. A striking observation was that prepartum L-tryptophan supplementation not only improved the milk production but also the quality compared to the control cows. In general, supplementation with L-tryptophan in the prepartum period can improve the postpartum reproduction and lactation performance of cows to some extent.

Obesity has emerged as a major risk factor for human health, exacerbated by aging and changes in dietary habits. It represents a significant health challenge, particularly for older people. While numerous studies have examined the effects of obesity and aging on fat metabolism independently, research on their combined effects is limited. In the present study, the protective action against white fat accumulation after a high-fat diet (HFD) exerted by exogenous melatonin, a circadian hormone endowed with antioxidant properties also involved in fat metabolism, was investigated in a mouse model. For this purpose, a battery of tests was applied before and after the dietary and melatonin treatments of the animals, including epididymal white adipose tissue (eWAT) histological evaluations, transcriptomic and lipidomic analyses, real-time PCR tests, immunofluorescence staining, Western blot, the appraisal of serum melatonin levels, and transmission electron microscopy. This study found that aged mice on a high-fat diet (HFD) showed increased lipid deposition, inflammation, and reduced antioxidant glutathione (GSH) levels compared to younger mice. Lipidomic and transcriptomic analyses revealed elevated triglycerides, diglycerides, ceramides, and cholesterol, along with decreased sphingomyelin and fatty acids in eWAT. The genes linked to inflammation, NF-κB signaling, autophagy, and lipid metabolism, particularly the melatonin and glutathione pathways, were significantly altered. The aged HFD mice also exhibited reduced melatonin levels in serum and eWAT. Melatonin supplementation reduced lipid deposition, increased melatonin and GSH levels, and upregulated AANAT and MTNR1A expression in eWAT, suggesting that melatonin alleviates eWAT damage via the MTNR1A pathway. It also suppressed inflammatory markers (e.g., TNF-α, NLRP3, NF-κB, IL-1β, and CEBPB) and preserved mitochondrial function through enhanced mitophagy. This study highlights how aging and HFD affect lipid metabolism and gene expression, offering potential intervention strategies. These findings provide important insights into the mechanisms of fat deposition associated with aging and a high-fat diet, suggesting potential intervention strategies.

Vitrification is a vital tool for the long-term preservation of animal genetic resources, yet cryoinjury—manifesting as oxidative stress, structural damage, and metabolic disorders—severely compromises its efficacy. Here, we investigated the protective effects of melatonin (MT) supplementation on the cryotolerance of mouse morulae. First, mouse morulae were assigned to four groups treated with vitrification and thawing media containing MT (0, 10⁻³, 10⁻⁵, and 10⁻⁷ M) to determine optimal MT concentration. Subsequently, embryos treated with the optimal MT concentration were evaluated for developmental competence, oxidative stress, apoptosis, and mitochondrial function. Furthermore, transcriptome sequencing was performed to elucidate MT-regulated molecular pathways. The results demonstrated that MT supplementation at 10⁻⁵ M significantly enhanced developmental competence, as evidenced by increased blastocyst rate, hatched blastocyst rate, total cell number and the inner cell mass (ICM)-to-total cell ratio compared to the MT-free group (p < 0.05). Consequently, embryo transfer outcomes showed higher live births and weaned pups in the 10⁻⁵ M MT group versus those in controls (p < 0.05), achieving levels comparable to fresh embryos (p > 0.05). Mechanistically, MT reversed cryoinjury-induced mitochondrial dysfunction by elevating membrane potential(MMP) and Adenosine Triphosphate(ATP) production while reducing Reactive Oxygen Species (ROS) accumulation (p < 0.05). Transcriptomic analysis further revealed that vitrification perturbed metabolic pathways, including amino acid/fatty acid degradation and glucose/pyruvate metabolism. MT downregulated cryoinjury-induced overexpression of Rela and Nfkb1, inhibiting excessive NF-κB activation and alleviating metabolic dysfunction. Additionally, MT restored expression of nucleotide synthesis genes (Ctps2, Nme4, Gmps, Nudt2, Ppat, Impdh2) critical for cell proliferation, and reversed downregulation of mitochondrial genes Sucla2 and Timm17a, confirming restoration of mitochondrial homeostasis. In conclusion, melatonin alleviates vitrification-induced cryoinjury by restoring mitochondrial function, which rescues nucleotide synthesis and partially reverses associated metabolic dysfunction. These findings advance MT-mediated cryoprotection and underscore its translational value for embryo cryopreservation in animal genetic resource conservation.