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.

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.

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.