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.

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.

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.

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.

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.

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.

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.

Melatonin promotes the development of sheep transgenic cloned embryos by protecting donor and recipient cells.

The yield efficiency of transgenic animal generation is relatively low[1]. To improve its efficiency has become a priority task for researchers[2]. Melatonin (N-acetyl-5-methoxytryptamine, MT) is a potent-free radical scavenger and antioxidant to protect mitochondria, lipids, protein and DNA from oxidative stress[3]. In this study, we observed that improving the quality of both donor and recipient cells by giving physiological concentration (10-7 M) of MT significantly increase the sheep transgenic embryo development in the in vitro condition. MT promotes the donor cell viability, proliferation, efficiency of monoclonal formation and the electrotransferring efficiency of fetal fibroblast cells (FFCs). The mechanistic exploration indicates that MT has the capacity for the synchronization of cell division cycle, reduction of cellular oxidative stress, apoptosis, and the increase of mitochondrial number and function. All of these render MT's ability to increase the efficiency of animal transgenic processes such as somatic cell nuclear transfer (SCNT) and electroporation. The outcomes are the increased cleavage rate and blastocyst rate of the transgenic sheep embryos after MT treatment. These beneficial effects of MT on transgenic embryo development are worth to be tested in the in vivo condition in the future.

Overexpression of ASMT likely enhances the resistance of transgenic sheep to brucellosis by influencing immune-related signaling pathways and gut microbiota.

Melatonin is a pleiotropic molecule with a variety of biological functions, which include its immunoregulatory action in mammals. Brucellosis is a worldwide endemic zoonotic disease caused by the Brucella, which not only causes huge economic losses for the livestock industry but also impacts human health. To target this problem, in current study, two marker-free transgenic sheep overexpressing melatonin synthetic enzyme ASMT (acetylserotonin O-methyltransferase) gene were generated and these melatonin enrich transgenic sheep were challenged by Brucella infection. The results showed that the serum melatonin concentration was significantly higher in transgenic sheep than that of wild type (726.92 ± 70.6074 vs 263.10 ± 34.60 pg/mL, P < .05). Brucella challenge test showed that two thirds (4/6) of the wild-type sheep had brucellosis, while none of the transgenic sheep were infected. Whole-blood RNA-seq results showed that differential expression genes (DEGs) were significantly enriched in natural killer cell-mediated cytotoxicity, phagosome, antigen processing, and presentation signaling pathways in overexpression sheep. The DEGs of toll-like receptors (TLRs) and NOD-like receptors (NLRs) families were verified by qPCR and it showed that TLR1, TLR2, TLR7, CD14, NAIP, and CXCL8 expression levels in overexpression sheep were significantly higher and NLRP1, NLRP3, and TNF expression levels were significantly lower than those of wild type. The rectal feces were subjected to 16S rDNA amplicon sequencing, and the microbial functional analysis showed that the transgenic sheep had significantly lower abundance of microbial genes related to infectious diseases compared to the wild type, indicating overexpression animals are likely more resistant to infectious diseases than wild type. Furthermore, exogenous melatonin treatment relieved brucellosis inflammation by upregulating anti-inflammatory cytokines IL-4 and downregulating pro-inflammatory IL-2, IL-6, and IFN-γ. Our preliminary results provide an informative reference for the study of the relationship between melatonin and brucellosis.

Effects of Melatonin on Dairy Herd Improvement (DHI) of Holstein Cow with High SCS.

Mastitis is a common disease in cows breeding. The milk quality will be significantly reduced with increased milk somatic cells, which often occurs in cows with mastitis. In this study, the influence of seasonal changes, age and lactation stages in the Dairy Herd Improvement (DHI) of cows was investigated. Then, the Dairy Herd Improvement (DHI) of cows with high somatic cell score (SCS) after melatonin treatment was systemically investigated. The results showed that melatonin significantly suppressed the milk somatic cell score under all of the tested conditions. The melatonin treatment also improved the milk nutritional value by reducing its fat but increasing its lactose and protein contents. The application of melatonin significantly improved the DHI. The beneficial effects of melatonin on DHI are likely attributed to the antioxidant and anti-inflammatory activities of melatonin.

Qualitative Identification of the Static Pull-In and Fundamental Frequency of One-Electrode MEMS Resonators.

This paper attempts to qualitatively identify the static pull-in position, pull-in voltage, and fundamental frequency of one-electrode microresonators from a physical perspective. During theoretical derivation, a generalized one-degree-of-freedom (1-DOF) model in nondimensional form derived using the differential quadrature method (DQM) is first introduced and then transformed for frequency normalization. Based on the deduced formulas, the upper and lower bounds of the static pull-in position and pull-in voltage are both deduced through mathematical proof. To distinguish the monotonic and nonmonotonic behavior of the fundamental frequency versus direct current (DC) voltage, a critical condition decided only by cubic stiffness is then determined. For the first time, two extreme static positions, as well as the corresponding fundamental frequencies and DC voltages to identify different frequency behaviors are derived, and their variations versus cubic stiffness are then discussed and verified. During the simulation process, a high-order DQM and COMSOL 2D model are both applied for numerical analyses. Guided by nondimensional results, typical behaviors with specific physical parameters are examined in detail. Results demonstrate that the curve tendencies between all the qualitative results and quantitative numerical simulations in dimensional form agree well with each other, implying the possibility of using 1-DOF model to qualitatively discuss physical parameters effects on the system statics and dynamics.

Vibration Identification of Folded-MEMS Comb Drive Resonators.

Natural frequency and frequency response are two important indicators for the performances of resonant microelectromechanical systems (MEMS) devices. This paper analytically and numerically investigates the vibration identification of the primary resonance of one type of folded-MEMS comb drive resonator. The governing equation of motion, considering structure and electrostatic nonlinearities, is firstly introduced. To overcome the shortcoming of frequency assumption in the literature, an improved theoretical solution procedure combined with the method of multiple scales and the homotopy concept is applied for primary resonance solutions in which frequency shift due to DC voltage is thoroughly considered. Through theoretical predictions and numerical results via the finite difference method and fourth-order Runge-Kutta simulation, we find that the primary frequency response actually includes low and high-energy branches when AC excitation is small enough. As AC excitation increases to a certain value, both branches intersect with each other. Then, based on the variation properties of frequency response branches, hardening and softening bending, and the ideal estimation of dynamic pull-in instability, a zoning diagram depicting extreme vibration amplitude versus DC voltage is then obtained that separates the dynamic response into five regions. Excellent agreements between the theoretical predictions and simulation results illustrate the effectiveness of the analyses.