Type of the study: Research article effect of melatonin on cryopreservation of Beijing you chicken (gallus gallus) spermatozoa.

Beijing You Chicken, a valuable local chicken breed from Beijing, China, was once listed as an endangered breed. From the point of view of conservation, the preservation of this breed is an important task for the local researchers. Semen cryopreservation is a popular method to maintain valuable species. However, during cryopreservation, semen is susceptible to oxidative damage. Melatonin is a potent antioxidant and free radical scavenger, so it has been selected to improve the efficiency of sperm cryopreservation. In this study, the chicken semen was treated with different concentrations of melatonin in the cryopreservation solution. The results showed that melatonin at concentrations of 10-3 M and 10-5 M significantly improved sperm progressive motility and total motility, respectively, compared to the control (P < 0.05). Melatonin at 10-3 M also significantly improved the plasma membrane and acrosome integrity of spermatozoa compared to the control. The mechanisms are that melatonin significantly reduces the level of ROS and preserves sperm mitochondrial membrane potential. Most importantly, the melatonin-treated cryopreserved chicken sperm after artificial insemination significantly increased the hatching rate of chicks compared to the control (p < 0.05). The results show that melatonin has a positive effect on the quality of the cryopreserved spermatozoa. These results provide the theoretical and practical basis for using melatonin to improve Beijing You Chicken conservation, and they may also be applicable to poultry as a whole.

Melatonin improves the vitrification of sheep morulae by modulating transcriptome.

Embryo vitrification technology is widely used in livestock production, but freezing injury has been a key factor hindering the efficiency of embryo production. There is an urgent need to further analyze the molecular mechanism of embryo damage by the vitrification process. In the study, morulae were collected from Hu sheep uterine horns after superovulation and sperm transfusion. Morulae were Cryotop vitrified and warmed. Nine morulae were in the vitrified control group (frozen), and seven morulae were vitrified and warmed with 10-5 M melatonin (melatonin). Eleven non-frozen morulae were used as controls (fresh). After warming, each embryo was sequenced separately for library construction and gene expression analysis. p < 0.05 was used to differentiate differentially expressed genes (DEG). The results showed that differentiated differentially expressed genes (DEG) in vitrified morulae were mainly enriched in protein kinase activity, adhesion processes, calcium signaling pathways and Wnt, PI3K/AKT, Ras, ErbB, and MAPK signaling pathways compared to controls. Importantly, melatonin treatment upregulated the expression of key pathways that increase the resistance of morulae against vitrification induced damage. These pathways include kinase activity pathway, ErbB, and PI3K/Akt signaling pathway. It is worth mentioning that melatonin upregulates the expression of XPA, which is a key transcription factor for DNA repair. In conclusion, vitrification affected the transcriptome of in vivo-derived Hu sheep morulae, and melatonin had a protective effect on the vitrification process. For the first time, the transcriptome profiles caused by vitrification and melatonin in sheep morulae were analyzed in single embryo level. These data obtained from the single embryo level provide an important molecular mechanism for further optimizing the cryopreservation of embryos or other cells.

The Effects of Mammary Gland ATIII Overexpression on the General Health of Dairy Goats and Their Anti-Inflammatory Response to LPS Stimulation.

Antithrombin III is an important anticoagulant factor with anti-inflammatory properties. However, few studies have explored its anti-inflammatory actions in ATIII overexpressed transgenic animals. In this study, the dairy goats with mammary overexpression of ATIII were used to investigate their general health, milk quality and particularly their response to inflammatory challenge. The results showed that transgenic goats have a normal phenotype regarding their physiological and biochemical parameters, including whole blood cells, serum protein levels, total cholesterol, urea nitrogen, uric acid, and total bilirubin, compared to the WT. In addition, the quality of milk also improved in transgenic animals compared to the WT, as indicated by the increased milk fat and dry matter content and the reduced somatic cell numbers. Under the stimulation of an LPS injection, the transgenic goats had elevated contents of IGA, IGM and superoxide dismutase SOD, and had reduced proinflammatory cytokine release, including IL-6, TNF-α and IFN-ß. A 16S rDNA sequencing analysis also showed that the transgenic animals had a similar compositions of gut microbiota to the WT goats under the stimulation of LPS injections. Mammary gland ATIII overexpression in dairy goats is a safe process, and it did not jeopardize the general health of the transgenic animals; moreover, the compositions of their gut microbiota also improved with the milk quality. The LPS stimulation study suggests that the increased ATIII expression may directly or indirectly suppress the inflammatory response to increase the resistance of transgenic animals to pathogen invasion. This will be explored in future studies.

A novel signaling transduction pathway of melatonin on lactose synthesis in cows via melatonin receptor 1 (MT1) and prolactin receptor (PRLR).

In the current study, we explored the relationship between melatonin and lactose synthesis in in vivo and in vitro conditions. We found that long-term melatonin feeding to the dairy cows significantly reduced the milk lactose content in a dose dependent manner. This lactose reduction was not associated with a negative energy balance, since melatonin treatment did not alter the fat, glucose, or protein metabolisms of the cows. To identify the potential molecular mechanisms, the cow's mammary epithelial cells were cultured for gene expression analysis. The results showed that the effect of melatonin on lactose reduction was mediated by its receptor MT1. MT1 activation downregulated the mRNA expression of the prolactin receptor gene (PRLR), which then suppressed the gene expression of SLC35B1. SLC35B1 is a galactose transporter and is responsible for the transportation of galactose to Golgi apparatus for lactose synthesis. Its suppression reduced the lactose synthesis and the milk lactose content. The discovery of this signal transduction pathway of melatonin on lactose synthesis provides a novel aspect of melatonin's effect on carbohydrate metabolism in cows and maybe also in other mammals, including humans.

Early Pregnancy Markers in the Serum of Ewes Identified via Proteomic and Metabolomic Analyses.

The diagnosis of ewes' pregnancy status at an early stage is an efficient way to enhance the reproductive output of sheep and allow producers to optimize production and management. The techniques of proteomics and metabolomics have been widely used to detect regulatory factors in various physiological processes of animals. The aim of this study is to explore the differential metabolites and proteins in the serum of pregnant and non-pregnant ewes by proteomics and metabolomics. The serum of ewes at 21, 28 and 33 days after artificial insemination (AI) were collected. The pregnancy stratus of the ewes was finally determined through ultrasound examination and then the ewes were grouped as Pregnant (n = 21) or N on-pregnant (n = 9). First, the serum samples from pregnant or non-pregnant ewes at 21 days after AI were selected for metabolomic analysis. It was found that the level of nine metabolites were upregulated and 20 metabolites were downregulated in the pregnant animals (p < 0.05). None of these differential metabolomes are suitable as markers of pregnancy due to their small foldchange. Next, the proteomes of serum from pregnant or non-pregnant ewes were evaluated. At 21 days after AI, the presence of 321 proteins were detected, and we found that the level of three proteins were upregulated and 11 proteins were downregulated in the serum of pregnant ewes (p < 0.05). The levels of serum amyloid A (SAA), afamin (AFM), serpin family A member 6 (SERPINA6) and immunoglobulin-like domain-containing protein between pregnant and non-pregnant ewes at 21-, 28- and 33-days post-AI were also analyzed via enzyme-linked immunosorbent assay (ELISA). The levels of SAA and AFM were significantly higher in pregnant ewes than in non-pregnant ewes, and could be used as markers for early pregnancy detection. Overall, our results show that SAA and AFM are potential biomarkers to determine the early pregnancy status of ewes.

Effects of melatonin on rumen microorganisms and methane production in dairy cow: results from in vitro and in vivo studies.

BACKGROUND: Methane (CH4) is a major greenhouse gas, and ruminants are one of the sources of CH4 which is produced by the rumen microbiota. Modification of the rumen microbiota compositions will impact the CH4 production. In this study, the effects of melatonin on methane production in cows were investigated both in the in vitro and in vivo studies. RESULTS: Melatonin treatment significantly reduced methane production in both studies. The cows treated with melatonin reduced methane emission from their respiration by approximately 50%. The potential mechanisms are multiple. First, melatonin lowers the volatile fatty acids (VFAs) production in rumen and reduces the raw material for CH4 synthesis. Second, melatonin not only reduces the abundance of Methanobacterium which are responsible for generating methane but also inhibits the populations of protozoa to break the symbiotic relationship between Methanobacterium and protozoa in rumen to further lowers the CH4 production. The reduced VFA production is not associated with food intake, and it seems also not to jeopardize the nutritional status of the cows. This was reflected by the increased milk lipid and protein contents in melatonin treated compared to the control cows. It is likely that the energy used to synthesize methane is saved to compensate the reduced VFA production. CONCLUSION: This study enlightens the potential mechanisms by which melatonin reduces rumen methane production in dairy cows. Considering the greenhouse effects of methane on global warming, these findings provide valuable information using different approaches to achieve low carbon dairy farming to reduce the methane emission. Video Abstract.

Investigation of SNP markers for the melatonin production trait in the Hu sheep with bulked segregant analysis.

BACKGROUND: As an important reproductive hormone, melatonin plays an important role in regulating the reproductive activities of sheep and other mammals. Hu sheep is a breed favoring for meat, with prolific traits. In order to explore the relationship between melatonin and reproductive function of Hu sheep, 7,694,759 SNPs were screened out through the whole genome sequencing analysis from high and low melatonin production Hu sheep. RESULTS: A total of 68,673 SNPs, involving in 1126 genes, were identified by ED association analysis. Correlation analysis of SNPs of AANAT/ASMT gene and MTNR1A/MTNR1B gene were carried out. The melatonin level of CG genotype 7,981,372 of AANAT, GA genotype 7,981,866 of ASMT and GG genotype 17,355,171 of MTNR1A were higher than the average melatonin level of 1.64 ng/mL. High melatonin Hu sheep appear to have better multiple reproductive performance. CONCLUSIONS: By using different methods, three SNPs which are associated with high melatonin production trait have been identified in Hu sheep. These 3 SNPs are located in melatonin synthetase AANAT/ASMT and receptor MTNR1A, respectively. Considering the positive association between melatonin production and reproductive performance in ruminants, these three SNPs can be served as the potential molecular markers for breading Hu sheep with the desirable reproductive traits.

The FSH-mTOR-CNP signaling axis initiates follicular antrum formation by regulating tight junction, ion pumps, and aquaporins.

The initial formation of the follicular antrum (iFFA) serves as a dividing line between gonadotropin-independent and gonadotropin-dependent folliculogenesis, enabling the follicle to sensitively respond to gonadotropins for its further development. However, the mechanism underlying iFFA remains elusive. Herein, we reported that iFFA is characterized by enhanced fluid absorption, energy consumption, secretion, and proliferation and shares a regulatory mechanism with blastula cavity formation. By use of bioinformatics analysis, follicular culture, RNA interference, and other techniques, we further demonstrated that the tight junction, ion pumps, and aquaporins are essential for follicular fluid accumulation during iFFA, as a deficiency of any one of these negatively impacts fluid accumulation and antrum formation. The intraovarian mammalian target of rapamycin-C-type natriuretic peptide pathway, activated by follicle-stimulating hormone, initiated iFFA by activating tight junction, ion pumps, and aquaporins. Building on this, we promoted iFFA by transiently activating mammalian target of rapamycin in cultured follicles and significantly increased oocyte yield. These findings represent a significant advancement in iFFA research, further enhancing our understanding of folliculogenesis in mammals.

Development and Validation of a 54K Genome-Wide Liquid SNP Chip Panel by Target Sequencing for Dairy Goat.

As an important genotyping platform, SNP chips are essential for implementing genomic selection. In this article, we introduced the development of a liquid SNP chip panel for dairy goats. This panel contains 54,188 SNPs based on genotyping by targeted sequencing (GBTS) technology. The source of SNPs in the panel were from the whole-genome resequencing of 110 dairy goats from three European and two Chinese indigenous dairy goat breeds. The performance of this liquid SNP chip panel was evaluated by genotyping 200 additional goats. Fifteen of them were randomly selected for whole-genome resequencing. The average capture ratio of the panel design loci was 98.41%, and the genotype concordance with resequencing reached 98.02%. We further used this chip panel to conduct genome-wide association studies (GWAS) to detect genetic loci that affect coat color in dairy goats. A single significant association signal for hair color was found on chromosome 8 at 31.52-35.02 Mb. The TYRP1 gene, which is associated with coat color in goats, was identified to be located at this genomic region (chromosome 8: 31,500,048-31,519,064). The emergence of high-precision and low-cost liquid microarrays will improve the analysis of genomics and breeding efficiency of dairy goats.

Melatonin alleviates lung injury in H1N1-infected mice by mast cell inactivation and cytokine storm suppression.

Influenza A virus (IAV) H1N1 infection is a constant threat to human health and it remains so due to the lack of an effective treatment. Since melatonin is a potent antioxidant and anti-inflammatory molecule with anti-viral action, in the present study we used melatonin to protect against H1N1 infection under in vitro and in vivo conditions. The death rate of the H1N1-infected mice was negatively associated with the nose and lung tissue local melatonin levels but not with serum melatonin concentrations. The H1N1-infected AANAT-/- melatonin-deficient mice had a significantly higher death rate than that of the WT mice and melatonin administration significantly reduced the death rate. All evidence confirmed the protective effects of melatonin against H1N1 infection. Further study identified that the mast cells were the primary targets of melatonin action, i.e., melatonin suppresses the mast cell activation caused by H1N1 infection. The molecular mechanisms involved melatonin down-regulation of gene expression for the HIF-1 pathway and inhibition of proinflammatory cytokine release from mast cells; this resulted in a reduction in the migration and activation of the macrophages and neutrophils in the lung tissue. This pathway was mediated by melatonin receptor 2 (MT2) since the MT2 specific antagonist 4P-PDOT significantly blocked the effects of melatonin on mast cell activation. Via targeting mast cells, melatonin suppressed apoptosis of alveolar epithelial cells and the lung injury caused by H1N1 infection. The findings provide a novel mechanism to protect against the H1N1-induced pulmonary injury, which may better facilitate the progress of new strategies to fight H1N1 infection or other IAV viral infections.

Genome-wide analysis for the melatonin trait associated genes and SNPs in dairy goat (Capra hircus) as the molecular breeding markers.

Previous studies have reported that the endogenous melatonin level is positively associated with the quality and yield of milk of cows. In the current study, a total of 34,921 SNPs involving 1,177 genes were identified in dairy goats by using the whole genome resequencing bulked segregant analysis (BSA) analysis. These SNPs have been used to match the melatonin levels of the dairy goats. Among them, 3 SNPs has been identified to significantly correlate with melatonin levels. These 3 SNPs include CC genotype 147316, GG genotype 147379 and CC genotype 1389193 which all locate in the exon regions of ASMT and MT2 genes. Dairy goats with these SNPs have approximately 5-fold-higher melatonin levels in milk and serum than the average melatonin level detected in the current goat population. If the melatonin level impacts the milk production in goats as in cows, the results strongly suggest that these 3 SNPs can serve as the molecular markers to select the goats having the improved milk quality and yield. This is a goal of our future study.

The Uterine Melatonergic Systems of AANAT and Melatonin Membrane Receptor 2 (MT2) Are Essential for Endometrial Receptivity and Early Implantation in Mice.

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.

Evaluate the developmental competence of human 8-cell embryos by single-cell RNA sequencing.

Abstract: The transition of maternal to zygotic gene expression regulation is critical for human preimplantation embryo development. In recent years, single-cell RNA sequencing (scRNA-seq) had been applied to detect the factors that regulate human oocyte maturation and early embryo development. Here, the evaluation of transcriptomes in single blastomere from the embryo collected from patients by scRNA-seq was performed. There were 20 blastomeres biopsied from 8-cell embryos of seven patients who received more than two ART cycles due to low embryo competence. Meanwhile, ten cells were collected from 8-cell embryos of four patients who received ART treatment due to male or tubal factors. The blastomeres were then evaluated using the previously established scRNA-seq method to determine the associations between their gene expression and developmental competence. The total number of genes detected in 8-cell embryos that failed to form blastocyst including maternal and zygotic mRNAs was reduced. There were 324 differently expressed genes detected among the 8-cell embryos including 65 genes that were significantly suppressed in the 8-cell embryos that failed to form blastocyst. Further analysis found these 8-cell embryos arrested at the cleavage stage due to the dysfunction of the cell cycle, DNA transcription activity, histone methylation, and cell division-related genes such as SMCO-1, ZNF271P,ZNF679, ASF1b, BEX3, DPPA2, and ORC4. The alterations of gene expression detected in human 8-cell embryos are tightly associated with its developmental competence and could be used as targets to enhance embryo development or parameters to predict the embryo's development outcomes. Lay summary: Many females are suffering infertility due to the failure of embryonic development at early stages due to unknown causes. At the very beginning of human embryo development, the embryos start to express its own genes, which should be achieved at 8-cell stage. In current research, we isolated one cell from 8-cell embryos and detected the gene expression at single-cell level. Then the remaining cells of these embryos were cultured to form blastocyst. Meanwhile, the data was analyzed according to the outcomes of embryo development. We detected 324 differently expressed genes between the 8-cell embryos that succeeded and failed to form blastocyst. Our research showed the association between the gene expression and the developmental competence of 8-cell embryos. The findings could be used to predict the embryo quality and potential therapy target to improve the efficiency of assisted reproductive techniques.

Melatonin Supplementation during the Late Gestational Stage Enhances Reproductive Performance of Sows by Regulating Fluid Shear Stress and Improving Placental Antioxidant Capacity.

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.

MDM2 antagonists promote CRISPR/Cas9-mediated precise genome editing in sheep primary cells.

CRISPR-Cas9-mediated genome editing in sheep is of great use in both agricultural and biomedical applications. While targeted gene knockout by CRISPR-Cas9 through non-homologous end joining (NHEJ) has worked efficiently, the knockin efficiency via homology-directed repair (HDR) remains lower, which severely hampers the application of precise genome editing in sheep. Here, in sheep fetal fibroblasts (SFFs), we optimized several key parameters that affect HDR, including homology arm (HA) length and the amount of double-stranded DNA (dsDNA) repair template; we also observed synchronization of SFFs in G2/M phase could increase HDR efficiency. Besides, we identified three potent small molecules, RITA, Nutlin3, and CTX1, inhibitors of p53-MDM2 interaction, that caused activation of the p53 pathway, resulting in distinct G2/M cell-cycle arrest in response to DNA damage and improved CRISPR-Cas9-mediated HDR efficiency by 1.43- to 4.28-fold in SFFs. Furthermore, we demonstrated that genetic knockout of p53 could inhibit HDR in SFFs by suppressing the expression of several key factors involved in the HDR pathway, such as BRCA1 and RAD51. Overall, this study offers an optimized strategy for the usage of dsDNA repair template, more importantly, the application of MDM2 antagonists provides a simple and efficient strategy to promote CRISPR/Cas9-mediated precise genome editing in sheep primary cells.

A rapid method to measure melatonin in biological fluids (milk and serum) with liquid chromatography-tandem mass spectrometry.

Since the global demanding of natural melatonin-enriched milk has been significantly increased in the populations of children and elder, the accurate and quick melatonin detection from milk is urgently required. Thus, the regular methods no longer satisfy this requirement. In the current study, we reported a novel method to extract melatonin from milk for liquid chromatography-tandem mass spectrometry melatonin detection. This novel method was to use cold methanol (-20℃) to precipitate proteins and fat in milk with one step to extract melatonin. Compared to the regular methods, it was devoid of procedures of sample drying, solid phase extraction and sample reconstitution. It could short the extraction time from the regularly 150 min to 60 min/per 24 milk samples. We believe that this novel method provides a possibility to detect large scale of milk samples in relatively short time with more efficiency and less cost compared to the regular method.

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.

The effects of rumen-protected tryptophan (RPT) on production performance and relevant hormones of dairy cows.

Tryptophan is an essential amino acid that cannot be synthesized in mammals. Therefore, the dietary supply of tryptophan is critical for the health and production performance (e.g., milk) of mammals. In the present study, 36 lactating Holstein cows were used, of which 24 cows were in the rumen-protected tryptophan (RPT) feeding groups with different doses at 14 g/d and 28 g/d, respectively and 12 cows were in the control group. This approach could avoid dietary tryptophan being degraded by the rumen microorganisms and improve its bioavailability for cows. The results showed that RPT increased milk protein percentage, milk protein yield, milk solid non-fat (SNF), and milk yield. In response to RPT treatment, the levels of melatonin (MT), prolactin (PRL), and insulin-like growth factor-1 (IGF-1) were significantly increased in the serum of cows compared to the controls. RPT feeding improved nutrient utilization efficiency and lactation performance of dairy cows, which enhanced the quality of milk.

Melatonin promotes the growth and development of lambs by increasing growth hormone and testosterone, targeting on apoptosis signaling pathway and intestinal microflora.

Melatonin is an indole-like neuroendocrine hormone. A large number of studies have shown that melatonin can improve production performance of ewes, but it is not clear in lambs. In this study, the growth and development of the 2-month-old lambs implanted with melatonin were monitored for 60 days. The results showed that the growth rate of body weight and body skew length of lambs with melatonin treatment were significantly improved compared to the controls. The similar results were also observed in red blood cell count, hematocrit, red blood cell volume distribution width, the levels of growth hormone, testosterone, immunoglobulin A, immunoglobulin M and albumin. In addition, the cross sectional area of muscle fibers and adipose cells of lambs with melatonin implantation were also significantly increased compared to the controls (P<0.05). To further explore the potential mechanisms, the muscle and adipose tissue were selected for transcriptome sequencing. KEGG enrichment results showed that melatonin regulated the expression of genes related to apoptotic signaling pathway in muscle and adipocytes. Since the intestinal microbiota are involved in the nutritional balance and animal growth, the 16SrRNA sequencing related to the intestinal microbiota was also performed. The data indicated that the structural differences of fecal microflora mainly occur in the pathways of Cardiovascular disease, Excretory system and Signaling molecules and interaction. In brief, melatonin promotes the growth and development of lambs. The potential mechanisms may be that melatonin increased the growth hormone and testosterone mediated apoptosis signaling pathway and regulated intestinal microbial flora. Our results provide valuable information for melatonin to improve the production of sheep husbandry in the future.