Gallus gallus) diets, optimized for maximum yield production vs. feed cost, have only begun to be assessed for impact on immune function. In order to study immune effects of dietary CP levels, different starter phase (day 1–14) diets were fed to 230 Ross 708 male broiler chicks randomly assigned at 1 day of age into two treatment groups. Group 1: Standard diet (STD) contained 3,000 kcal AMEn/kg energy and 23.78% CP; and Group 2: Reduced crude protein diet (RCP) contained 3,000 kcal AMEn/kg energy and 21.23% CP. From day 15–35 a common standard grower/finisher diet  was allocated to both groups. Zymosan, a glycan derived from yeast cell walls that binds to TLR 2 and Dectin-1, was used for intra-abdominal challenge. Results demonstrated that a reduced crude protein starter diet (21.23 vs. 23.78% CP) between age 1–14, while maintaining the same levels of metabolizable energy and essential amino acids, did not affect broilers growth performance or lymphoid organ weights (P > 0.05). Interestingly, basal leukocyte levels in the RCP group significantly (P < 0.01) increased in the blood compartment at d35 in the unchallenged birds. Significant enhancements to leukocyte infiltration into the abdominal cavity were also detected post-immune challenge with zymosan . Post-challenge levels of TNF-α, IL-1β, and CXCL8 gene expression cells collected from the abdominal cavity were not affected by the diets (P > 0.05). Moreover, dietary treatments did not influence percentage of ROS producing cells in the abdominal cavity (P > 0.05). To our best knowledge, this is the first study that reports the impacts of reduced crude protein diet on the innate immune response of poultry to an acute inflammation model in the abdominal cavity. Overall, our results highlight that reduced crude protein diets can be used without negatively impacting broiler performance and may enhance the capacity of broilers to recruit leukocytes upon infection.Crude protein (CP) levels in commercial broiler ( Broilers have been exposed to intense genetic selection for enhanced performance traits, primarily for fast growth and efficiency of feed utilization, and thus may have sustained detrimental effects on the immune system , 2. AlreAnother important factor which modulates immune function, while interacting with genetic make-up, is the composition of diet. Dietary protein sources are often a high expense component of poultry diets. As animals require specific amino acids, formulations requiring specific amino acids often lead to crude protein (CP) levels that are overly costly and result in excess nitrogen being excreted as an environmental pollutant . The indin vivo intra-peritoneal/intra-abdominal injection of zymosan has already added to our understanding of immune status in comparative animal studies including fish, pigs, mice, and chickens  were randomly assigned at 1 day of age into two dietary treatment groups: standard crude protein (STD) and reduced crude protein (RCP). In order to maximize data quality and minimize stress-associated impact, parallel pens were used for immunological and performance measurements. Eight pen replicates were used in total, with six pens of 15 birds (three pens per treatment) for growth performance measurements  and two pens of 70 birds (one pen per treatment) for immunological parameters . Dietary treatment groups were set up in an alternating pen pattern within the barn. All birds were housed in the Poultry Research Facility of the Department of Agricultural, Food and Nutritional Sciences at the University of Alberta. All animals were maintained according to the guidelines specified by the Canadian Council on Animal Care, and protocols were approved by the University of Alberta Animal Care and Use Committee.ad libitum throughout the experiment. From d 1–14 two different starter diet with different CP levels were allocated, with amino acid ratios balanced based on Evonik recommendations (AMINOChick®). The RCP diet contained 3,000 kcal AMEn /kg and 21.23% CP, while the STD contained 3,000 kcal AMEn /kg and 23.78% CP. From d 15–35 the same standard grower/finisher diet  was allocated to all groups. The feed formulation and nutritional composition of the starter (1–14 days of age) and grower (15–35 days of age) diets are shown in Diets  and water were provided Body weight was measured weekly for each pen. Feed intake was determined as the difference between the amount of feed offered and the amount unconsumed in starter and grower phase. The daily feed intake (DFI) was calculated by dividing each pen's consumed feed on starter and grower phase by actual total number of birds. The feed conversion ratio (FCR: g feed/g body weight gain) was calculated by dividing daily feed intake by daily body weight gain.The femur bone, thymus, bursa of Fabricius and spleen weight were measured at 7, 14, 28, and 35 d of age from five birds per group. These organs and the corresponding chicks were weighed, and organs relative weight was expressed as a percentage of body weight (BW).Saccharomyces cerevisiae  was resuspended in 500 ml of PBS−/−  and administered through injection into the abdominal cavity to induce inflammation response. Birds that did not receive an injection at 0 h were used as the control group. This injection dose allowed the establishment of self-resolving conditions, where early induction and late resolution phases of acute inflammation could be observed.Induction of an intra-abdominal acute inflammation was derived from a custom protocol previously validated in our laboratory . BrieflyWhole peripheral blood samples were taken by wing vein venipuncture. In order to determine the short- and long-term immune impacts of diets, total leukocyte numbers, heterophil, monocyte/macrophage, and lymphocyte populations were measured at 0, 4, and 12 h after zymosan intra-abdominal injections on d 14 and 35 in the bone marrow and abdominal cavity. Bone marrow leukocytes were obtained after femur collection, where bones were flushed with 20 ml of sterile RPMI-1640 medium. Red blood cells were removed from blood and bone marrow samples through treatment with a red blood cell lysis buffer  (1:10 ratio). Intra-abdominal leukocytes were recovered by injecting 10 and 20 ml of RPMI-1640 medium on d 14 and 35, respectively. Cells were harvested and maintained at 4°C. Chickens that did not receive an injection were used as a negative control (0 h birds). Blood total leukocytes were counted in a hemocytometer chamber using Natt and Herrick's solution to obtain a 1:200 blood dilution .5 cells) from peripheral blood, bone marrow, and intra-abdominal were spun onto glass slides at 55 g for 6 min at room temperature using a cytocentrifuge (Thermo Scientific ™ Cytospin ™ 4) and stained using the Hema3 staining set (Fisher Scientific) according to the manufacturer's specifications. Images were generated using a DM1000 microscope  and a brightfield 100× objective .Leukocytes  and analyzed using INSPIRE software .ROS production was determined based on the CellROX oxidation procedure (Molecular Probes). Briefly, 1 × 10−ΔΔCt method. The primer sequences for each gene are shown in Gene expression was performed as previously described , 20. Bripost hoc tests were also performed for analyzing the differences within the treatments and time after intra-abdominal challenge. Bursa of Fabricius, thymus, femur, spleen, body weight, feed intake, and feed conversion ratio data were analyzed by t-test. Statistics with P < 0.05 were considered significant.Evaluation of significance was based on two-way analysis of variance (ANOVA) analyses with time point and treatment as main effects, using Sidak's multiple comparison tests in Prism 8 software . Additional Tukey's P > 0.05; Reduced dietary crude protein in the starter phase of broilers did not significantly affect body weight gain, feed intake, or feed conversion ratio in either the starter phase or beyond until 35 days of age , evident at both d 14 and d 35 (P < 0.01). The higher leukocyte migration in the RCP group at d 14 correlated with higher basal levels of naïve mature neutrophils within the bone marrow storage pool , as well as significantly higher rates of leukocyte egression . This was likely associated with higher leukocyte egression from this hematopoietic compartment in route to the abdominal challenge site . In contrast, higher levels of TNF-α expression (P < 0.01). Enhanced levels of TNF-α expression are consistent with the higher levels of leukocyte infiltration described above in RCP group after intra-abdominal challenge.The early phase of acute inflammation is manifested by a multifaceted functionality that involves the release of lipid mediators such as prostaglandin E2, leukotriene C4, thromboxane A2, chemokines such as CXCL8, and pro-inflammatory cytokines such as TNF- α, IL-1 β, IL-6, IL-17, which ultimately contribute to the recruitment of leukocytes to the site of inflammation . TNF-α apression  were detin vivo immune challenge using zymosan as a fungal mimic. This was observed during the starter phase up to day 14 of age, after which all birds were fed a common standard crude protein grower diet. Additionally, the total leukocyte levels in the blood at day 35 in unchallenged treatments were higher in the RCP compared to STD groups. These changes persisting into the grower period may point to epigenetic changes affecting immune development in response to the changes in nutrition occurring directly post-hatch during the developmental starter phase. It remains to be investigated whether the observed immune developmental changes are a direct consequence of the dietary protein levels, or reflect differences in the availability of free vs. protein bound amino acids between the diets, and/or due to the differences in starch:lipid ratios of the diets as a result of the changes to raw ingredient inclusion to reduce CP. While no difference in the inflammatory mediators or ROS production could be observed, it is hypothesized that the time points for observing these changes potentially occurred earlier than the first samplings. Future research could involve additional earlier time points, as well as additional inflammatory markers to further understand the drivers and subsequent changes in immune activity, in response to the dietary CP levels in the starter phase. The industry would also benefit from further research, to investigate how the immune changes occurring from RCP in the starter phase may benefit vaccination strategies, disease resistance to different kinds of field relevant pathogens, or growth performance recovery post-challenge. In conclusion, this study provides exciting new evidence to support the potential benefits of starter phase RCP diets for the development and response of the immune system in broilers.In summary, moderate reductions in dietary CP in the starter phase of broilers did not significantly affect growth performance or relative organ weights in either the starter phase or beyond until 35 days of age. This was to be expected as diets were formulated to meet all standard ileal digestible essential amino acid requirements in optimal ratios. However, compared to the STD treatment, RCP in the starter phase significantly increased leukocyte recruitment to the abdominal cavity in response to an The datasets generated for this study are available on request to the corresponding author.All animals were maintained according to the guidelines specified by the Canadian Council on Animal Care, and protocols were approved by the University of Alberta Animal Care and Use Committee.MK, JW, RW, VN, and DB conceived and designed the study. MK and WH performed the experiments. MK analyzed and interpreted the data, prepared the figures, and drafted the manuscript. All authors participated in reviewing the manuscript and approved the final version of the manuscript. All authors contributed to the article and approved the submitted version.RW and VN were employed by Evonik Nutrition & Care GmbH. This work was supported by the Evonik Nutrition & Care GmbH grant RES0037933. The funder contributed to study design and manuscript review. The funder was not involved in collection, analysis, or interpretation of data. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


ALPL gene encoding the ectoenzyme Tissue-nonspecific alkaline phosphatase (TNAP). Considering the establishment of zebrafish (Danio rerio) as a new model organism for HPP, the aim of the study was the spatial and temporal analysis of alpl expression in embryos and adult brains. Additionally, we determined functional consequences of Tnap inhibition on neural and skeletal development in zebrafish. We show that expression of alpl is present during embryonic stages and in adult neuronal tissues. Analyses of enzyme function reveal zones of pronounced Tnap-activity within the telencephalon and the mesencephalon. Treatment of zebrafish embryos with chemical Tnap inhibitors followed by axonal and cartilage/mineralized tissue staining imply functional consequences of Tnap deficiency on neuronal and skeletal development. Based on the results from neuronal and skeletal tissue analyses, which demonstrate an evolutionary conserved role of this enzyme, we consider zebrafish as a promising species for modeling HPP in order to discover new potential therapy strategies in the long-term.Hypophosphatasia (HPP) is a rare genetic disease with diverse symptoms and a heterogeneous severity of onset with underlying mutations in the  ALPL gene, which encodes the ectoenzyme tissue-nonspecific alkaline phosphatase (TNAP). The disease is classified due to the age of diagnosis and the severity of onset1. Clinical HPP manifestations show a broad spectrum of severity depending on the degree of ALPL deficiency, ranging from stillbirth or perinatal lethality to severe or moderate manifestations in childhood, adolescence and adulthood to the odonto-form of HPP that is restricted to teeth1. Apart from frequently occurring mineralization defects of bones and teeth, premature loss of deciduous teeth and craniosynostosis presuppose additional therapy for affected patients1. Moreover, neurological symptoms such as epileptic seizures, depression, sleep- or anxiety-disorders further hamper HPP-patients’ quality of life2. The prevalence for severe HPP forms has been estimated 1:300.000 and 1:6,370 for moderate forms in the European population3. To date 409 mutations (including 71.4% missense mutations) localized in the human ALPL gene have been listed in the database that is provided by E. Mornet . The mode of inheritance for HPP is either autosomal dominant or recessive, whereas the latter is common for severe cases and both traits have been described for mild onsets of the disease3. Additionally, dominant negative mutations are frequently reported in the context of HPP and its underlying ALPL mutations3. Genotype–phenotype correlations have been published, however cases have been described where same genotype leads to different severity of symptoms5.Hypophosphatasia (HPP) is a rare hereditary disease with a diversity of systemic manifestations including a predominant skeletal phenotype. The genetic cause of HPP are mutations in the 1. Remarkably, comparative studies have already shown that the predominant TNAP transcript variant in bones is the same as in brain tissues6, which implies a common mode of TNAP function in both tissues. TNAP’s most important biochemical function in bones and teeth is providing the basis for mineralization processes. The enzyme enables hydroxyapatite crystallization in bone via catalyzing the dephosphorylation of mineralization inhibitors such as inorganic pyrophosphate (PPi) and phosphorylated osteopontin7. In the nervous system, two predominant mechanisms, the availability of vitamin B6 and alterations in purinergic signaling9, significantly influence the outcome of the disease. In case of decreased TNAP-activity, pyridoxal-5-phosphate (PLP), which is the transportable form of vitamin B6, accumulates within the serum and cannot be redistributed into the brain without dephosphorylation via TNAP. PLP is an essential enzymatic co-factor within central neurotransmitter synthesis pathways in the brain and the lack of PLP results in neurological impairment due to limited biochemical conversion8. Additionally, TNAP enzyme supports the development and maintenance of synapse functionality and is involved in the outgrowth and myelination of neurites12. Localization of TNAP within primates’ brains has been described as distinct patterns within layer 4 and 5 of the cortex14 and has been detected in the retina across a number of different vertebrate species15. In mice, TNAP localization was detected predominantly in endothelial cells, primordial germ cells, pioneer growth cones, and neural precursors16. Akp2 (the murine version of the human ALPL gene) knockout mice display reduced serum levels of alkaline phosphatase, elevated substrate levels, impaired bone mineralization, and frequently die from epileptic seizures18. Due to the early lethal phenotype in mice and the lack of supplementary in vivo studies within other vertebrates, our knowledge about TNAP’s interconnected functions within bone and neuronal tissues is still scarce.Different tissues express different alkaline phosphatase (AP) isoforms and HPP patients’ phenotypes can greatly differ in the responsiveness between tissuesDanio rerio) has become an important vertebrate model organism for bone20 and brain research21. Along with other advantages, including low cost and easy housing of the animals, the possibility to investigate transgenic reporter lines by non-invasive in vivo monitoring and perform large scale screenings, zebrafish offer a wide repertoire of different approaches for investigation of bone and brain development24. Furthermore, basic molecular pathways for vertebrate development, like bone development, are evolutionarily conserved and share common properties with other species26. Consequently, a rising number of in vivo models for musculoskeletal diseases have been established in recent years29 and even the molecular background of complex diseases, such as craniosynostosis, has been faithfully reproduced in a zebrafish model32. Despite the analysis of mineralization and skeletal issues, feasible strategies for the investigation of complex neurological diseases, such as anxiety disorder and depression, have already been successfully modeled within the zebrafish37. Comparison of multiple neurological functions linked to the amygdala is one example, which has been attributed to being relevant for anxiety in a wide number of different vertebrate species35. Furthermore, novel genetic and visualization techniques primarily established in zebrafish enable detailed cellular resolution of gene expression during brain development and open up the opportunity to image neural activity in freely behaving larvae39.Due to a number of biological properties, the zebrafish  were bred and maintained as previously described44 in the aquatic facilities of the Biocenter of the Julius-Maximilians-University Würzburg, Germany according to FELASA guidelines. In short, adult fish are kept at a mean temperature of 24–26 °C in 10 l glass and 2.5 l plastic tanks, while embryos younger than 120 h post-fertilization (hpf) are raised at a temperature of 28.5 °C in an incubator. A daily light cycle of 10 h dark/14 h light is maintained. Preconditioned reverse osmosis water with adjusted conductivity 800–1,100 µS/cm, pH7.0 and stable water hardness is used. A food combination of Artemia nauplii and GEMMA Micro Food  is standard.Zebrafish (AB/TU (ZDB-GENO-010924-10), AB/AB (ZDB-GENO-960809-7) and nac/tra . Embryos were staged by morphological characteristics according to literature42. The denomination “hpf” indicates hours post fertilization at 28.5 °C.All experimental procedures were performed according to the guidelines of the German animal welfare law and approved by the local government . Used zebrafish strains: AB/AB strain). For cDNA synthesis, 1 µg RNA was transcribed into cDNA and further analyzed in a ViiA7 Real-Time PCR System . Each group and primer sample was analyzed via qPCR in triplicates on a single plate utilizing HOT FIREPol Eva Green Mix Plus (Solis BioDyne). Data analysis was performed via QuantStudio Real-Time PCS Software v1.1 by ΔΔCt method. Muscle tissue served as reference sample for relative comparison. Amplification of gapdh and eef1a1l1 was used as endogenous cDNA controls (housekeeping genes). Used primer pairs and targeted regions are noted in Table For qPCR experiments, RNA was extracted from adult zebrafish organs . Samples were stored at − 80 °C until experimental analyses. For protein extraction, samples were disrupted in cold PBS/protease inhibitor  at a ratio of 25:1 using micro pestles and sonication. The samples were then centrifuged at 10.000 g at 4 °C for 10 min and the supernatant was transferred into a new tube. The protein content was determined using the RotiQuant solution  following manufacturer’s instructions. The alkaline phosphatase activity was measured according to previously published protocols11. Briefly, 2 µg protein per sample were diluted in 100 µl of cold PBS/PI containing various concentrations  of the TNAP-inhibitor levamisole, and finally 100 µl CSPD tricyclo decan}-4-yl)phenyl phosphate) ready-to-use reagent  were added. The reaction was performed in white 96-well-plates with a clear bottom at 37 °C for 5 min. For measuring the luminescence, which was resulting from the degradation of CSPD reagent by TNAP enzyme, the Orion II Microplate Luminometer  was used.Organs of adult fish were extracted and instantly frozen in liquid nitrogen . We used an alpl cDNA fragment of 434 bp size to synthesize specific anti-sense RNA probes. Sense probes were synthesized as negative control for each anti-sense probe and were used under the same reaction conditions. Additional details on experimental set-up is given in the supplementary methods.ISH was performed according to standard protocols47.TNAP ISH was performed using the same TNAP-specific riboprobes as described for the embryonal stages subsequently followed by immunofluorescence (IF). PCNA-specific staining was performed using the primary antibody in a dilution of 1:200  and anti-mouse IgG Alexa Fluor 488  secondary antibody in a dilution of 1:1,000. Finally, the brains were embedded using the JB-4 embedding kit  according to manufacturer’s instruction and the brain samples were sectioned in 4 µm slices. Additional details on experimental set-up is given in the supplementary methods. Brain regions were sketched, identified and named according to Wullimann et al.48. The primary monoclonal mouse antibody IgG2b 6-11B-1  was used in the dilution of 1:100. The anti-mouse IgG Alexa Fluor 488  secondary antibody was used in a dilution of 1:1,000 in PBST/Triton X-100/10% sheep serum (Sigma-Aldrich/Merck) including Hoechst 33,342 nuclear counterstain .Immunohistochemistry against acetylated α-Tubulin was performed according to previously published protocolsAB/AB wildtype fish, according to the manufacturer’s instructions with certain modifications. Specificity control of ELF staining was performed by TNAP inhibitor application and is shown in Fig. ELF 97 Phosphatase Substrate (Thermo Fisher Scientific), which is converted into a fluorescence precipitate by active AP enzymes, was used on adult brain cryosections of The embryos were collected directly after fertilization, the chorion was punctured using a glass needle, and treatment with the respective inhibitors was performed in a final concentration of 0.5, 1, and 10 mM in 0.3× Danieau’s medium for levamisole  and in a concentration of 10, 50, and 100 µM for MLS-0038949 . Incubations were performed until 24 and 48 hpf for IF and 120 hpf for mineralized tissue and cartilage staining (change of medium after 48 h). For the MLS-0038949 treatment, DMSO served as a solvent control, in accordance with the volume of the highest MLS-0038949 concentration used . The chorion was punctured prior to treatment in order to guarantee a proper and reproducible incubation with the respective inhibitor.49. Zebrafish were fixated with 4% PFA/PBS for 2–4 h and afterwards incubated with 50% EtOH for 10 min. Double staining was performed on a shaker overnight prior to washing with ddH2O. Afterwards, the samples were bleached for 20 min in 3% H2O2/PBS and subsequently washed with glycerol solutions containing KOH ).Cartilage and mineralized structures were visualized by acid-free double staining with Alcian-blue and Alizarin-red according to previously published protocols50. To visualize ELF 97 staining a UV/DAPI longpass fluorescence filter set was used. Images were processed by using ImageJ/Fiji (https://fiji.sc/), CorelDraw Graphics Suite x8 software (Corel Corporation), and EnfuseGUI (https://software.bergmark.com/enfuseGUI/Main.html).Depending on the experiment, images were acquired either with a Leica S8 APO Stereomicroscope (whole embryos), a Zeiss Imager A1 (ISH), or a Nikon A1 + Laser scanning confocal microscope (IF). In order to visualize the NBT/BCIP fluorescence signal on ISH sections, a corresponding near infrared filter was usedALPL gene encoding the ectoenzyme TNAP  is widely conserved across a wide number of animal species . For providing consistent differentiation between species as well as between gene and protein, we used the respective conventional denominations in our manuscript. The gene name is ALPL in human, Alpl in mouse, and alpl in zebrafish, whereas the protein is called TNAP in human and mouse but Tnap in zebrafish. Detailed information on the human, murine, and zebrafish orthologues are listed in Fig. The alpl gene with the human and mouse sequences revealed an identity of 61% and 63%, respectively. In contrast to many other genes, the alpl gene is not duplicated within the zebrafish genome. Interestingly, the peptide alignment resulted in 67% overall identity to mouse and humans. Moreover, the alignment showed a remarkable high conservation level of 79% identity and 86% similarity in amino acid sequences of the zebrafish and the human alkaline phosphatase domain. Additional synteny analysis further revealed that genes located in close proximity to the respective ALPL, Alpl, and alpl locus were showing a high level of genetic conservation between human, mouse, and zebrafish on the respective chromosomes 1, 4, or 11  within the midbrain-hindbrain boundary (MHB) region of the neural keel , the pectoral fin, the heart field, and the tail bud  were detected within the eyes (RQ: 146.1), followed by the whole brain (RQ: 49.4), and the skull samples  values of ata Fig. .alpl mRNA but high Tnap-activity values were detected in skin samples, while high mRNA expression but low Tnap-activity were detected in whole brain samples. Apart from these, the alpl expression and Tnap-activity were corresponding to each other.The qPCR data was complemented with measurements of enzymatic Tnap-activity within several organs isolated from adult zebrafish , cortex , ventral mesencephalon , and rhombencephalon  as schematically depicted in Fig. gapdh and eef1a1l1; Fig. alpl in the telencephalon (RQ: 10.04), followed by the cortex (RQ: 7.20), the mesencephalon (RQ: 4.48), and finally the rhombencephalon (RQ: 1.38). Additionally, samples from eyes were analyzed as a positive control. Subsequently, ISH was performed on whole adult zebrafish brains  and the commissura tecti .Additional JB-4 sections were prepared at different levels of the mesencephalon, a second brain region showing high ion Fig. . Six difion Fig. A and impalpl expression and Tnap-activity pattern in the adult zebrafish brain revealed, that the highest prevalence of Tnap is localized to the telencephalon, here especially to the dorsal and lateral regions. Additionally, alpl expression is, among others, prevalent in the hypothalamus, the thalamus, and interestingly also in cells of the ventricular regions, which are important zones for adult neurogenesis. Our own, novel data have been compared to previously published expression patterns in primates, mice and zebrafish in Table In summary, the analysis of the In order to investigate a possible influence of Tnap inhibition on the early development of the nervous system and the skeleton in zebrafish, embryos were treated with the Tnap-specific inhibitor levamisole at different concentrations . After morphological analysis of the embryos within the first 48 h Fig. A, functiLevamisole at low doses (0.5 and 1 mM) had no or only a slight impact on the regular morphological development of the treated zebrafish embryos at 24 and 48 hpf. Abnormal developmental effects, aberrations to untreated/DMSO control embryos and increased lethality were concentration dependent and increased in intensity with higher levamisole concentration Fig. A. The anFurthermore, cartilage and mineralized tissue staining of levamisole treated embryos revealed aberrant craniofacial development see Fig. C. Interealpl Splice and ATG Morpholinos for knockdown experiments and for recapitulating the indicated influence of Tnap during the development of the nervous system and mineralization of the zebrafish  instead of the tissue-nonspecific isoform of the AP . Additionally, the highest alpl expression levels along with strong AP-activity can be observed in the eyes, more precisely in the lens and the retina, which seems to be conserved between different vertebrate species and is in line with previously published literature15. However, no specific symptoms concerning the visual performance of HPP patients have been described so far and the exact role of TNAP/Tnap in the visual system and potential compensatory mechanisms remain to be unraveled in future experiments.Furthermore, our experiments affirm that alpl was additionally analyzed in dissected and in sections of adult zebrafish brains. The results show that alpl expression is prevalent in the telencephalon , the tectum opticum, and in the hypothalamus . Moreover, we can frequently detect alpl expression in ventricular zones of the brain (e.g. TeIV and TeV). The alpl expression pattern implies restricted Tnap-activity in the zebrafish brain, but staining for ELF 97 shows rather wide areas of Tnap-activity in the telencephalon, in the hypothalamus, and in the torus longitudinalis. Proteolytic shedding processes might lead to cleavage and spreading of active Tnap enzyme within brain compartments and subsequently result in tissue-wide activity. Release of TNAP from the cellular membrane via degradation of its phospholipase mediated GPI-anchor has already been reported and might be one molecular mechanism for this observation54. Nevertheless, it is unclear to what extent those processes might lead to the observed discrepancy between the different staining methods. Another interesting observation are strong ISH signals within the dorsomedial optic tract (DOT), a white matter structure comprising predominantly of retinal ganglion cell axons. Localization of TNAP to axons of cultured hippocampal neurons has been previously reported and TNAP-activity is crucial for correct axonal growth by ATP degradation10. Another explanation for the discrepancy between ISH and ELF 97 data may further result from different sensitivity thresholds of the respective staining method.For detailed investigation of the nervous system related symptoms of HPP and for confirmation of qPCR and CSPD-assay results, the endogenous expression of alpl expressing domains in the brain sections. In total, 16 proliferative zones have already been described in the adult zebrafish brain, which are predominantly located at the ventricular surface, and incorporate neuronal stem cells55. Compared to zebrafish, the mammalian brain shows only limited capability of adult neurogenesis. Novel formation of neurons in adult brains is only possible in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus in humans56. Interestingly, Tnap-activity is indeed located in the ventricular and SVZ of mice including zones of adult neurogenesis57.The proliferation marker PCNA is partly co-localized or localized in close proximity to the ALPL signals can be verified in specific brain regions of different mammals and in layers across the neocortex , for example in layer 4 of the primary visual and somatosensory cortices. Our zebrafish results are comparable to expression patterns observed in mammals and indicate that alpl transcripts can not only be detected during embryogenesis, but also in specific brain structures of adult animals. Generally, ALPL expression levels are higher during vertebrate development compared to mature organs, tissues , or the complete animal58. The results of our own study and literature data on the ALPL, Alpl, and alpl expression in adult brains are summarized in the Table Prominent alpl and inhibition of TNAP-activity at very early embryonic stages of the zebrafish implies that the enzyme plays an important role during initial steps of development. This assumption is further promoted by patients suffering from genetically determined severe TNAP deficiency and by homozygous Alpl knockout mice, which often die very shortly after birth66. The inhibition of Tnap-activity, either with chemical components (levamisole and MLS-0038949) or by knockdown of alpl expression using Morpholinos, impairs the development of cartilage/bone as well as the nervous system in zebrafish67. Considering the known side effects of levamisole, like ion channel inhibition68, the investigation of MLS-0038949 and alpl knockdown was indispensable in order to confirm the results. Our studies with the alternative inhibitor MLS-0038949 and with two alpl Morpholinos affirm the levamisole results to the greatest extent. However, we observe differences between the two inhibitors, as MLS-0038949 is effective at much lower doses and displays a stronger effect on axonal growth even at 10 µM. Levamisole needs to be prevalent in higher doses for effective blocking and shows more prominent effects on bone development over time. Our results clearly show that both inhibitors and Morpholino knockdown are able to repress Tnap-activity, but differ in their influence on neuronal and skeletal development. These results imply different modes of TNAP functional reduction and might point to different biochemical consequences. TNAP/Tnap’s promoting role in hydroxyapatite crystallization is mediated by diminishing the amount of prevalent mineralization inhibitor PPi in the extracellular space during in vitro osteogenic differentiation69. While axonal outgrowth is initiated by preventing the activation of the purinergic receptor of P2X710. Overall, the observed effects of Tnap inhibition on early neuronal and bone development indicate a rather broad spectrum of Tnap efficacy, including effects on dephosphorylation of general substrates, such as PPi, ATP/ADP/AMP, and phosphorylated osteopontin70. Despite the low level of interference of the Splice Morpholino with the wildtype gene product and general concerns with Morpholino usage, the data are supporting our results gained with the Tnap inhibitors levamisole and MLS-0038949. However, we have to point out that, deviant of the Stanier et al. guidelines, no mutant lines have been generated yet, which would be essential for a complete characterization of the phenotype. The establishment of transgenic zebrafish lines has to be done in future research projects. Nevertheless, we think that we can trust the inhibitor and Morpholino data as they are confirming an influence of decreased Tnap-activity on mineralization processes and neuronal development, which is additionally going along with the phenotype of HPP patients.The expression of alpl expression in zebrafish as well as first experiments on functional consequences of decreased alpl expression/Tnap inhibition on the embryonic development of bones and the nervous system pave the way for consecutive analysis of transgenic zebrafish lines in the future. These will help to further elucidate so far unsolved molecular mechanisms that are underlying certain HPP symptoms. One unsolved symptom to be investigated is how the paradox situation of premature suture fusion in the developing skull can be reconciled with an environment of hypomineralization in the rest of the body. Moreover, future studies have to investigate how neurological symptoms, like depression or anxiety disorders, can indeed robustly be linked to alpl deficiency in single brain regions or to specific neurons72. An important role of TNAP/Tnap during the processes of synaptogenesis, myelination, neurotransmitter metabolism, and axonal outgrowth has already been proclaimed, but has not been investigated in zebrafish13. Therefore, those cellular developmental functions definitely have to be considered for establishing a complete picture of TNAP’s function in the nervous system of zebrafish by applying cell-type-specific methods. Generally, our results of the embryonic and adult alpl expression analysis in zebrafish suggest a possible correlation to the pathology of the rare disease HPP and open up new perspectives concerning the neurological consequences of TNAP/Tnap dysfunctions.In summary, our study identifies the zebrafish as a suitable model for the rare disease HPP. The analysis of endogenous Supplementary file1


Ever-increasing demands of industrial manufacturing regarding mechanical properties require the development of novel alloys designed towards the respective manufacturing process. Here, we consider wire arc additive manufacturing. To this end, Al alloys with additions of Zn, Mg and Cu have been designed considering the requirements of good mechanical properties and limited hot cracking susceptibility. The samples were produced using the cold metal transfer pulse advanced (CMT-PADV) technique, known for its ability to produce lower porosity parts with smaller grain size. After material simulations to determine the optimal heat treatment, the samples were solution heat treated, quenched and aged to enhance their mechanical performance. Chemical analysis, mechanical properties and microstructure evolution were evaluated using optical light microscopy, scanning electron microscopy, energy dispersive X-ray spectroscopy, X-ray fluorescence analysis and X-ray radiography, as well as tensile, fatigue and hardness tests. The objective of this research was to evaluate in detail the mechanical properties and microstructure of the newly designed high-performance Al–Zn-based alloy before and after ageing heat treatment. The only defects found in the parts built under optimised conditions were small dispersed porosities, without any visible cracks or lack of fusion. Furthermore, the mechanical properties are superior to those of commercial 7xxx alloys and remarkably independent of the testing direction . The presented analyses are very promising regarding additive manufacturing of high-strength aluminium alloys. Additive manufacturing (AM) is a manufacturing technology which has been evolving at an enormous rate in the last 30 years. AM uses 3D CAD models to build parts by adding material successively, layer by layer, allowing the production of complex geometric shapes and topology optimised parts. The additive manufacturing of metals, which has become an area of extensive research work, is transforming many industrial sectors by reducing component lead time and material waste. Many different industries using metallic materials, such as aerospace, automotive, tooling, and health care, among others, are already taking advantage of AM ,2. Never2 phase) evolving from the Zn and Mg Guinier–Preston (GP) zones [2CuMg (S-Phase) [Due to the high strength-to-weight ratio and ductility at room temperature, Al–Zn–Mg–Cu alloys are used in the automotive, aerospace, aircraft and competitive sport industries ,5,6,7. IP) zones ,9,10. ThS-Phase) ,12,13. 2, T (Al2Mg3Zn3) and S (Al2CuMg)-phase precipitates [The ageing of Al–Zn–Mg–Cu alloys from room temperature to relatively low ageing temperatures is accompanied by the generation of GP zones with an approximately spherical shape . With inipitates ,16,17.153Si2) instead of the more harmful β-phases (Al5FeSi) [The effect of alloying elements on welding additive processability and mechanical properties of Al–Zn–Mg–Cu alloys have been studied in different research works ,19,20. FAl5FeSi) ,21,22,23Al5FeSi) . FurtherAl5FeSi) ,26. The Among the manifold additive manufacturing techniques, technologies using metal powder can produce parts with high accuracy and comparably low deposition rates, which renders them more suitable to produce small parts with complex geometry . When thThe aim of the present work was to understand the influence of the wire arc process and new alloying system on the mechanical properties of the produced parts before and after the heat treatment, including detailed microstructural characterisation.The experimental alloy composition was cast using vertical continuous casting. The resultant chemical composition is given in 3 (l × w × h) and an R equal to 5 mm for the corners were manufactured  and a robot arm  using CMT pulse advanced (PADV). Rectangular-shaped parts of approximately 170 × 30 × 120 mmfactured . A waterFor further analyses, three different material conditions were investigated in detail: (i) the as-built (AB) material condition; (ii) the T6 material condition, which was obtained by a solution heat treatment at 470 °C for 5 h, followed by rapid cooling in water and a subsequent ageing treatment at 120 °C for 24 h; and (iii) the T73 material condition, which was obtained by a solution heat treatment at 470 °C for 5 h, followed by rapid cooling in water and a subsequent two-stage ageing treatment at 120 °C for 24 h, and then 160 °C for 24 h.Tensile test specimens were extracted from the samples in the three different material conditions, in both transverse and longitudinal directions a. SubsizFatigue test specimens were extracted from the T73 tempered material condition in the longitudinal and transverse direction. The test conditions and specimen geometry  were in N, Abingdon, UK).The transverse parts in the various conditions mentioned above were cold mounted, grinded with SiC paper and polished with diamond paste. Microhardness measurements were performed with a hardness tester , using the Vickers method, with a load of 9.81 N and indentation time of 15 s. These measurements were performed in the centreline of the specimens along its height . Microstructure analysis was performed by optical light microscopy  and stereoscopy . Specimens were electrochemically etched with Barkers Reagent (5 mL HBF4 (48%) + 200 mL water) using 15 V for 90–240 s. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) results were obtained from the samples in as polished condition using a JSM–6500F microscope  fitted with an EDS detector  . First, 2 was considered. In most cases, five images were evaluated for each condition.In the grain measurement, the standard ASTM E112–2013 was used as a reference to establish the procedure. The number of grains in the specimens and respective areas were calculated using ImageJ software based on the visual counting performed in the etched micrographs. The images evaluated were of 200x or 500x magnification depending on the grain size. The ratio of the grains per area were then calculated based on the numbers of grain counted in a known area. To compare all the results, the average number of grains per 10⁶ μmMatCalc software  version 6.02 with the corresponding thermodynamic, mobility and physical databases for Al alloys was used for material simulations. The precipitation sequence during ageing was modelled in a simplified manner by considering Cu–Mg clusters and AlMgZn GP zones, nucleating directly from the FCC Al precipitation domain (bulk nucleation sites), while T-phase and S-phase precipitates were formed by a transformation of clusters and GP zones. Ageing treatments with various heat treatments were simulated, taking the annealed material at 500 °C as the initial material condition for precipitation kinetics simulations. For the validation of the results observed, experimentally, samples from the wire arc deposition process were solution heat treated to a peak temperature of 500 °C for 20 min. After quenching, three samples were subjected to an ageing process: (i) at 90 °C for 24 h, (ii) at 140 °C for 24 h and (iii) pre-aged at 90 °C for 24 h and aged at 140 °C for 24 h afterwards. Brinell hardness measurements were conducted in various time intervals during ageing. The chemical composition considered for the simulation was based on the chemical analysis of the alloy obtained after deposition. In many instances, the limiting factor with regard to manufacturing is the capability of the alloys to be shaped into components. Consequently, there is a continuous demand to improve existing alloys, as well as to design new ones, in order to meet the requirements of various processing technologies. In the design of the alloy investigated in the present work, engineering properties such as weldability, hot-tearing susceptibility as well as resulting mechanical properties were in focus as detailed by Schnall et al. . The cheThe absence of visible cracking, major porosity and no macroscopic shrinkage is a good indication of the process stability and the apparent good weldability of the newly designed alloy. Visual inspection of the specimens’ surfaces  suggestsThe arc’s colour during deposition appeared cyan, qualitatively suggesting the burn off of volatile chemical species. In Based on the macrographic results , it can A constant level of hardness is seen along the height of the walls, where the average values found for samples in the AB, T6 and T73 conditions are 103, 131 and 143 HV, respectively . The conWhen evaluating the micrographs of the deposited samples , it can The pore volumetric fraction analysis shows that this parameter increases along the samples’ height from the bottom to the top . This reWhen evaluating the number of grains in the different regions  of the sample in the T6 and T73 conditions , it can In order to estimate the effects of the various heat treatments on the mechanical properties, MatCalc simulations were performed as described in subchapter 2. The maximum yield strength within the simulated time frame of 500 h was observed for ageing at 90 °C, when the material approaches a value of 380 MPa, as can be seen in For validation of the simulation results, Brinell hardness tests were conducted and compared to the simulated yield strength for three different conditions . For theConcerning the deposition direction in the AB, T6 and T73 conditions, the tensile strength and elongation are lower in transverse specimens, indicating a certain degree of anisotropy . It shouIn the AB, T6 and T73 conditions, the tensile strength and the fracture elongation for standard and subsize specimens are similar, indicating homogenous mechanical properties along the height of the produced samples, as indicated by the mechanical characteristics of the analysed specimens taken in transverse direction .Due to the limited amount of material available, fatigue testing was performed on specimens in the T73 condition only. This testing was intended to compare the material behaviour in the transverse and longitudinal directions, thus to obtain some indications on the performance of the manufacturing process, rather than fully characterise the fatigue behaviour of the material. The fatigue test results exhibited a considerable scatter for both deposition directions , particuThe main fracture initiation site feature evidenced in almost all specimens analysed was porosity . Post-teSome intergranular brittleness was also observed, possibly as a result of minor oscillations of deposition conditions leading to some fatigue endurance reduction. This intergranular fracture mode seemed more frequent in perpendicular specimens than in longitudinal ones .Besides intergranular brittleness, the initiation region and earlier crack growth was often associated with cleavage fracture of grains shifting in a later propagation stage and final unstable fracture to dimpled ductile fracture. Again, porosity was often observed in the propagation path as well as intergranular decohesion, more frequent in the perpendicular specimens .Despite the differences in the fracture behaviour, the material shows an almost identical behaviour in both longitudinal and transverse directions, which precludes potential for future use in mechanical parts. Comparison of fatigue data is difficult given the large variation of heat treatments, materials and testing conditions. The small number of fatigue tests performed in the present work also contribute to impair such comparisons. Nevertheless, the present results evidence the processability of 7xxx aluminium alloys by wire arc additive manufacturing with remarkable mechanical properties ,47,48.Wire arc additive manufacturing of the Al5–Mg3–Zn–Cu alloy resulted in a component with exceptionally high mechanical strength when compared to other Al alloys. In fact, the achieved mechanical properties are even superior to the values available for many commercial 7xxx alloys. Furthermore, the heat treatment simulations indicate that even higher mechanical properties can be achieved using optimised ageing treatments.Concerning defects, the manufactured parts only exhibit few dispersed porosities and volumetric pore fraction changes along the height of the sample. The volumetric pore fraction is smaller in the bottom and bigger in the top region, while the grains are smaller in the bottom and bigger in the upper region. Both observations are in line with an increasing mean component temperature with an increasing height. Equiaxed and elongated grains are seen along the sample—a typical feature of wire arc processed aluminium. It can be noted that smaller equiaxed grains are seen in the interlayer region, followed by elongated grains and a zone of coarser grains with mostly equiaxed grains.The fatigue results showed a high scatter caused mainly by residual porosity, which was the main feature associated with failure initiation and eventual risk of intergranular brittleness.2 or T (Al2Mg3Zn3) phases, respectively) from GP zones.The high strength reached under 60 h of ageing time can be attributed to the two-stage ageing treatment, which promoted full formation of precipitation and growth of very fine precipitates of the η′ or T′-phase (semi-coherent, metastable precursors of the equilibrium MgZnThe hardness measurements performed on the aged samples deposited by the wire arc process confirmed the simulation trends and the advantages of the two-stage ageing treatment as a processing method for this material.From the performed analyses of the novel Al–Zn–Mg–Cu alloy with improved processability, the following major conclusions can be drawn:This study demonstrates that the wire arc additive manufacturing of a novel 7xxx alloy is not only feasible but results in very good mechanical properties following ensuing heat treatments.


Although epidural spinal stimulation (ESS) results in promising therapeutic effects in individuals with spinal cord injury (SCI), its potential to generate functional motor recovery varies between individuals and remains largely unclear. However, both preclinical and clinical studies indicate the capacity of electrical and pharmacological interventions to synergistically increase the engagement of spinal sensorimotor networks and regain motor function after SCI. This study explored whether selective pharmacological antagonism of the adenosine A1 receptor subtype synergizes with ESS, thereby increasing motor response. We hypothesized that selective pharmacological antagonism of A1 receptors during ESS would produce facilitatory effects in spinal sensorimotor networks detected as an increased amplitude of spinally-evoked motor potentials and sustained duration of ESS induced activity. Terminal experiments were performed in adult rats using trains of stereotyped pulses at 40 Hz delivered at L5 with the local administration to the cord of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). We demonstrated that ESS combined with the blockage of A1 receptors increased the magnitude of the endogenous modulation and postponed the decay of responses that occur during ESS alone. Although DPCPX significantly increased the yield of repetitive stimulation in intact spinal cords, the effects of A1 antagonism on motor evoked responses after an acute spinal transection was not detected. These studies support the future investigation of the optimal dosage, methods of delivery, and systemic effects of the synergistic application of A1 antagonists and spinal stimulation in the intact and injured spinal cord. Further, when electrically stimulated, neural networks can be reorganized to generate responses and motor activities previously thought to be permanently lost due to paralysis  directly to the spinal cord of adult rats during supra-motor threshold ESS and assessed the neuromodulatory effects of DPCPX on spinally-evoked motor potentials. Although A2 receptors have been considered functionally marginal for the spinal sensorimotor circuits  Guide for the care and use of laboratory animals and the European Union directive on animal experimentation (2010/63/EU). Adult Long-Evans rats  were anesthetized by intraperitoneal (i.p.) administration of ketamine (100 mg/Kg) and xylazine (5 mg/Kg) mix and terminal electrophysiological recordings were obtained. During each experiment, toe pinches were performed periodically to assess whether the anesthetic level was maintained. If animals exhibited a reflex, a booster of ketamine was administered, as needed. Additionally, animals were kept under anesthesia over a heating pad (37°C) throughout each experiment. Finally, at the end of all experiments (2–3 h), animals were euthanized by COn = 8) and rats with transected spinal cords (n = 6). After collecting baseline recordings to serve as internal controls for each animal, the intact cord group was further split to explore the effects of DPCPX (n = 5) or DMPX (n = 3) applications. The transected group was also further divided into those exposed to DPCPX  and sham control (n = 1). For the sham control animal, trains of pulses were regularly supplied as in the treated groups, albeit without the presence of any substances. The tests demonstrated the stability of baseline responses for the entire length of a recording session after acute spinal transection (50 min). This feature is illustrated in P = 0.490, Friedman repeated-measures analysis of variance (ANOVA) on ranks, n = 3].A cartoon schematizing the experimental design of the study is provided in The prototypic experimental design is schematized in ®, ADInstruments Pvt. Ltd., Bella Vista, NSW, Australia).EMG signals from bilateral TA and left GM muscles were derived from belly muscles using 13 mm paired subdermal needle electrodes  inserted through the shank skin. A ground electrode was placed subcutaneously on the left forearm. Before starting the experiment, proper placement of electrodes was confirmed by the motion artifact generated when tapping the muscle. EMG recordings were amplified  using a differential AC amplifier  and subsequently digitalized at 40 kHz 12/Th13 and the complete removal of lumbar (L)1 lamina, two multistrand, Teflon-coated stainless-steel wires  was slid through the small opening at Th12/Th13 up to L2. A small notch (0.5–1.0 mm) in each wire was deprived of insulation at L1 vertebral level  to expose the conductor and form the electrodes, which were then placed on each side of the dorsal cord . The two wires were used as cathode and anode for bipolar stimulation. Single or repetitive current pulses (duration 100 μs) were supplied to the cord using a DS8R constant-current stimulator . Trains of 2,000 stereotyped rectangular pulses at 40 Hz  were delivered every 5 min.An acute complete transection of the cord was performed in five animals at the upper thoracic level (vertebrae Th8 to Th11) using a pair of iridectomy scissors. The resulting gap was inspected by another expert surgeon and filled by a small cotton ball for the entire duration of the experiment. DPCPX  and DMPX  were locally applied to the cord by diluting each drug in 500 μl of the saline medium. In four out of 14 experiments, before adding the two pharmacological agents, the dura was opened with a longitudinal incision along the midline of the entire spinal segment L5 (leaving the dura intact under the two electrodes). Given that no difference in drug effect was found when the dura remained intact compared to when it was not intact, data were pooled from both groups. This observation confirmed previous reports on the full permeability of the spinal dura to the substances used in the current study  and Clampfit® version 10.3 software . The amplitude of all consecutive responses evoked by each train was plotted for each muscle as time-course graphs. In the animals in which simultaneous recordings were obtained from bilateral TA and left GM, all muscles showed equal responses as for latency  and peak to peak amplitude . The amplitude of suprathreshold responses in bilateral TAs and left GMs did not differ across muscle groups or among tested animals. Therefore, values were first averaged among different muscles in response to the same train, then averaged among repetitions from the same experiment, and finally pooled among different animals for statistical comparison between treatments. The sum of amplitude responses was calculated from raw values to obtain the mean cumulative amplitude for each treatment. To identify whether spontaneous variations in the peaks of responses followed a rhythmic pattern of modulation, the procedure schematized in ® . The analysis adopted a default rectangular windowing function, with data segments not overlapping, window length set at the largest value fitting within the data segments to be processed, and the first spectral bin of the periodogram excluded from RMS measurements. The magnitude of the resulting spectrum is the summed power of all rhythm frequencies. This statistical tool quantifies any increase in frequency and/or amplitude of EMG evoked-responses, expressed as a complex rhythm composed of multiple harmonics.Amplitude and latency (defined at the first deflection of baseline) of all EMG responses were determined using Labchart® version 3.5 software  to compare the mean ± SD of different experimental conditions. All parametric values were analyzed using a two-tailed Student’s t-test (paired or unpaired) to compare two groups of data or a one-way ANOVA for more than two groups. Nonparametric comparisons were performed using the Mann–Whitney rank-sum test (unpaired) and Wilcoxon signed-rank test (paired) for two groups and Kruskal–Wallis ANOVA for more than two groups. Friedman repeated-measures ANOVA on ranks was performed for multiple comparisons. Multiple comparisons were followed by post hoc tests (Dunn’s Method). Results reached significance when P < 0.05.Data are indicated as mean ± standard deviation (SD) values and n refers to the number of rats used. The normality of data distribution was determined based on a Kolmogorov–Smirnov normality test. Statistical analysis was performed using SigmaStatP = 0.132, paired t-test, t = 1.703; n = 8). To identify if the spontaneous variation of responses followed a particular rhythmic pattern of modulation, the mean time-course of peaks were analyzed using power spectral analysis (n = 5). A rhythmic component centered around 1.41 ± 0.69 Hz and with an amplitude of 0.0017 ± 0.0011 mV/Hz2 described the presence of a spontaneous rhythmic pattern of modulation with a power spectrum magnitude, expressed as RMS equal to 0.20 ± 0.13, n = 5.In eight intact animals, electrical stimulation of the cord (intensity = 0.71 ± 0.26 mA) evoked control suprathreshold responses with a mean latency of 5.46 ± 0.77 ms and a mean peak to peak amplitude equal to 0.57 ± 0.45 mV. In analysis ; n = 5. These data indicate that continuous epidural stimulation with a train of pulses at 40 Hz produces EMG responses that are intrinsically modulated in amplitude by a spontaneous oscillatory rhythm of a lower frequency than the one supplied by the stimulating pattern.P = 0.080, paired t-test, t = −2.331; n = 5). P = 0.046, paired t-test, t = −2.861; n = 5). P = 0.044, Kruskal–Wallis one-way ANOVA on ranks followed by post hoc multiple comparisons vs. DPCPX 1 μM group with Dunn’s method, n = 5, 5, 5, 5, 4). Surprisingly, the first motor response evoked by a suprathreshold electrical stimulus applied to the spinal cord  was unaffected by 5 μM of DPCPX, as were the mean latency (5.44 ± 0.85 ms) and the mean peak to peak amplitude (0.50 ± 0.23 mV) of pooled data from five experiments. In the presence of DPCPX (5 μM), the average time-course of peak amplitudes showed a trend of higher responses compared to control during the first 300 pulses from the beginning of stimulation . Moreover, with respect to control, multiple motor responses of higher amplitude appeared for the entire duration of the stimulation protocol . To evaluate whether the selective pharmacological blockade of the A1 adenosine receptor subtype affects spinal cord stimulation, trains of 2,000 pulses (40 Hz) were serially delivered during increasing concentrations of the selective antagonist DPCPX (1–100 μM). The application of DPCPX at any dosage did not cause spontaneous activity in the muscles. The lowest concentration tested (1 μM) did not affect the cumulative amplitude of responses  and with an unchanged amplitude of 0.0036 ± 0.0031 mV/Hz2 . However, following the administration of DPCPX (5 μM), the frequency spectrum was more populated by multiple harmonics at higher frequencies . Moreover, in the presence of DPCPX (5 μM), the power spectrum magnitude of rhythmic modulation significantly increased, as expressed by RMS equal to 0.32 ± 0.21 .The power spectrum of the spontaneous amplitude of oscillations throughout the entire protocol demonstrated the presence of a rhythmic pattern of modulation with a frequency component equal to 1.29 ± 0.51 Hz, as reported in control .In contrast, the selective A2 antagonist, DMPX, did not affect the amplitude of spinally-evoked EMG responses at any of the concentrations tested  and mean amplitude . In P = 0.005, t-test, t = 3.810; n = 5) and were completely abolished after approximately the 800th pulse (~25 s of stimulation) under continuous stimulation at the same intensity .In five animals, an acute transection of the spinal cord at the low thoracic level did not affect motor responses to single epidural pulses applied below the lesion site . When compared to intact control responses, spinal reflexes elicited by single suprathreshold stimuli, after acute injury, showed unchanged mean latency , albeit of negligible amplitude .The power spectrum of the mean time-course was largely suppressed  from stimulation onset, with the amplitude scarcely modulated by the endogenous rhythmic pattern.P = 0.953; Kruskal–Wallis one-way ANOVA on Ranks; n = 5), following transection when compared to baseline responses.In the current study, we reported that DPCPX augmented the amplitude of repetitive responses in intact animals. Moreover, we indicated that, after an acute spinal transection, the motor output elicited by a train of pulses was dramatically reduced. To verify whether the selective antagonism of A1 receptors could rescue motor reflexes immediately following spinal transection, increasing concentrations of DPCPX were applied to the lumbosacral cord below the level of transection. However, no differences were revealed in the cumulative amplitude of reflexes, even at higher DPCPX concentrations (50–100 μM; Despite the promise of ESS in subjects with SCI (Angeli et al., In the clinic, repetitive ESS with a train of electrical pulses has been applied at different frequencies to elicit distinct types of motor recovery in select groups of young adults with chronic and complete SCI (Barolat et al., It remains to be seen whether the optimal frequency of stimulation should be tuned to the intrinsic physiologic pattern of activity to maximize the effectiveness of neuromodulation.In particular, it is yet to be explored whether spinal stimulation implemented by delivering trains of stimuli within the range shown to be physiologically effective and containing the principal components of the intrinsic physiological patterns of modulation (Gerasimenko et al., During training sessions in the presence of stimulation in humans, the motor output cannot be sustained at the same level and has been noticed to decline with the duration of the training and stimulation (Rejc et al., The facilitatory effect of the A1 antagonist on the amplitude of motor output illustrated in the current study is explained by two tentative mechanisms. First, DPCPX selectively blocks inhibitory presynaptic receptors on primary afferents to maximize input delivery. Second, the antagonism of postsynaptic A1 receptors depressing the activity of spinal networks (Witts et al., In the present study, although DPCPX largely increased the yield of repetitive stimulation in intact cords, after a spinal transection the selective antagonism of A1 receptors appeared to be ineffective. This can be in line with the contrasting and paradoxical effects of adenosine agonists and antagonists reported in various neurodegenerative conditions (Stone et al., per se, to generate any coordinated motor activity from hindlimb muscles. Rather, it can be speculated that the endogenous pattern of modulation represents an intrinsic and rhythmic tone able to set the subthreshold excitability of propriospinal circuits (Taccola et al., in vitro (Lampl and Yarom, In the current study, we further explore how the amplitude of motor responses evoked by repetitive pulses applied to a rodent spinal cord is modulated by the endogenous spinal rhythm (Taccola et al., The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Institutional Animal Care and Use Committee at Houston Methodist Research Institute.GT conceptualized the study. GT, DS, PJH, BHS, and MKH performed experiments. GT, RA, and DS analyzed data. GT prepared figures. GT and DS drafted the manuscript. All authors revised the manuscript and approved the final version. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Due to the negative public opinion and welfare issues related to surgical castration, gradual introduction of alternatives like entire males and immunocastrates is taking place. Despite several economical and welfare advantages that the alternatives offer, numerous issues , as well as their interactions with rearing and breeding (genetic) factors remain unanswered. Therefore, the focus of this study was to investigate the possibility of using different commercial sire lines in three male sex categories  and to compare their carcass traits, meat quality and boar taint compounds. A significant effect of terminal sire line and/or male category together with their interaction was observed for most of the investigated carcass and meat quality traits. The observed interaction should be taken into consideration when deciding on the production of a specific male category.n = 60), surgical castrates  and entire males . TSL affected most of the carcass and meat quality traits, together with the androstenone concentration. At the same time, MC had a significant effect on fat thickness, ham circumference, drip loss, cooking loss and androstenone and skatole concentrations. A significant interaction effect was observed for carcass length and ham circumference, as well as for most of the measured meat quality traits ). Among the three investigated sire lines, immunocastration was shown to be most beneficial for fatteners sired by the TSL C. However, if deciding to raise EM, fatteners from the TSL B are recommended in terms of carcass and meat quality, although strategies for avoiding boar taint in their carcasses must be taken into consideration.The aim of the research was to investigate the influence of terminal sire line (TSL) and male category (MC) on carcass and meat quality of commercial fatteners. The study was performed on 180 pigs originating from three terminal sire lines: A (Pietrain × Large White), B (pure Pietrain), and C (Pietrain × Duroc × Large White), being assigned to three groups according to MC: immunocastrates (IC,  Surgical castration causes severe pain to animals, thus raising a welfare concern for the pig industry  and a neIn EM, the occurrence of unfavourable organoleptic pork traits, the so-called “boar taint” presents the key issue. Boar taint is described as offensive urine-, sweat- and faecal-like odour and/or taste, attributed to substances skatole and androstenone that are deposited in fat tissue of uncastrated animals . BesidesFew effective strategies are available in attempts to avoid the boar taint in EM. One of the possible solutions includes genomic selection ,9, whichCompared to EM, immunocastrated pigs exhibit certain advantages , such as better feed efficiency and reduced aggressiveness  without The comparison of meat quality and carcass characteristics between different pig male categories  has been described by numerous authors , but is,Therefore, the aim of the present study was to investigate the influence of different male categories in relation to their genetic background  on carcass traits, meat quality and boar taint compounds of fatteners originating from three different male categories reared in an intensive production system.The experimental protocol was approved by the Bioethics Committee of the Faculty of Agrobiotechnical Sciences Osijek , and all procedures were performed in accordance with the Croatian Animal Welfare Act and other legal acts regulating animal husbandry and welfare.The study was performed on 180 fatteners originating from three different commercial terminal sire lines (TSL): A (Pietrain × Large White), B (pure Pietrain) and C (Pietrain × Duroc × Large White). The animals used in the experiment were selected from 30 litters farrowed within two weeks. The sows used were all of the identical genotype (Landrace).® , as recommended by the manufacturer. During the experiment, the animals were group-penned (10 pigs/pen) according to their designated sex category.Within each litter, two pigs were surgically castrated , two were designated for immunocastration  and two were raised as entire males . Surgical castration was performed during the first week of life. In the IC group, pigs were vaccinated by applying two doses of Improvac2 of available floor space per animal. The floor was partly concrete slatted and partly fully solid, with the fresh straw added every day to cover most of the fully solid area. In addition to natural light, artificial light was applied for 13 h per day. The temperature was 34 °C at birth and was gradually lowered to 13 °C before slaughter.The size of the pens was 3.60 m × 2.40 m, allowing 0.86 mIn order to avoid stress and aggressiveness, pigs were kept in the same pens from weaning to the end of the experiment. All pigs were kept under the same housing conditions with ad libitum access to food and water.During the trial, the animals were fed the following commercial diets : SO-1 (13.58 MJ ME/kg and 17.36% crude protein) from weaning to approximately 30 kg live weight (LW); ST-2 (13.26 MJ ME/kg and 16.05% crude protein) from 30 to approximately 70 kg LW; and ST-3 (12.95 MJ ME/kg and 14.06% crude protein) from 70 kg LW to the end of the experiment.2.After reaching the average weight of 110 kg, the animals were transported to a nearby commercial abattoir where they were slaughtered according to the conventional procedure that included stunning with COos pubis to cranial edge of 1st rib), ham length (from the anterior edge of the symphysis ossis pubis to the hock joint) and the ham circumference at its widest point were measured. Lean meat percentage (LMP), muscle thickness (M) and fat thickness (S) were determined by the two-point method approved in Croatia [After 24 h of cooling, the carcass length  and final (pH24) pH values were measured 45 min and 24 h post mortem in longissimus dorsi (LD) muscle using Mettler MP 120-B portable pH meter . Drip loss was determined using the EZ-DripLoss method [Initial  and ISO 13299 . All panellists were able to detect both androstenone in the pure form and preselected tainted samples of meat.The intensity of the boar taint was assessed in IC and EM by a trained six-member sensory panel based on ISO 4121  were taken from head meat and salivary glands according to German directive AVVLmHyg (2011)  and werep < 0.05 was classified as a significant difference. The obtained data were analysed using the Dell Statistica (2015) [The obtained data were analysed by factorial ANOVA  with log transformation of androstenone and skatole concentrations prior to the analyses. The influence of the TSL, male category (MC) and their interaction (TSL * MC) was determined by the Tukey HSD test, where a (2015) , while ta (2015)  in the Ra (2015) .p < 0.01) several carcass traits of investigated pigs, except carcass weight, muscle thickness and ham length (p < 0.01) than those originating from TSL A and lower LMP (p < 0.01) than fatteners originating from both TSL A and B. In addition, the progeny of TSL C sires had significantly longer carcasses compared to progeny of the TSL A (p < 0.01). Progenies of the TSL B did not differ with the other two lines in this trait. The hams of pigs originating from TSL A had significantly lower circumference than the pigs from TSL B and C (p < 0.01). The results of the current study regarding the backfat thickness and LMP support the earlier findings of Škrlep et al. [TSL influenced  and ham circumference (p = 0.016). The highest fatness was recorded in SC pigs followed by IC, and EM pigs had the lowest (p < 0.01). Similar results were also reported by a number of other similar studies [p = 0.473), agreeing with the results of Škrlep et al. (2010 and 2011) [A significant effect of MC was observed for fat depth  ,49, and nd 2011) , who didIn the present study, no significant differences were detected in LMP between the MC of investigated pigs, which is not in line with the literature ,51, indiThe carcasses of investigated male categories did not differ in their length. These results are also in disagreement with the findings of other studies where EM and IC pigs exhibited longer carcasses than SC ,55. It sp < 0.001) and ham circumference (p = 0.010), as presented in A significant interaction of TSL and MC was observed for carcass length  is not easy to explain. There are not many studies dealing with the interaction between the male category and breed on carcass traits . As to tGenerally, crosses sired with Pietrain are superior in lean meat percentage (LMP) and its indicators (fat depth and muscle thickness), whilst Duroc sires produce progeny characterized by superior growth rate, but with lower LMP in the carcasses. Due to higher intramuscular fat (IMF), their meat is therefore more appropriate for the production of high-quality products like dry-cured hams ,60,61. Tp < 0.05) between different sire lines in pH45 were observed ; however, no differences could be found between different male categories for this trait. In agreement with the results of this study, the results of the three meta-analyses showed no or very small difference between different male categories in the case of muscle pH, indicating this trait to be without actual practical importance in regard to sex categories [p < 0.001) between the TSL*MC on initial pH values  were found between different genotypes and male categories for pH24 (p = 0.006) between TSL and MC was observed , as suggested by Forrest [Although no significant differences  of TSL (higher in C compared to A and B), and , as well as their interaction (p = 0.002) on drip loss values was observed.It is known that water holding capacity strongly influences pork sensory traits, especially colour, flavour, and tenderness, apart from its economical and nutritional influence . In the EM originating from the TSL B exhibited the highest drip loss, while the lowest drip loss was observed in SC animals originating from the TSL C. Within TSL A, IC exhibited higher drip loss values than EM and SC animals, although no significant differences were observed between different male categories in this trait. In TSL C, SC exhibited the lowest drip values, followed by EM and IC animals A.In their review on meat quality of different male sex categories, Škrlep et al. (2020)  argued tp < 0.001), with EM having the highest values for this trait (p = 0.013), where meat from fatteners originating from TSL B was tenderer than the meat from fatteners originating from TSL A. The difference is difficult to relate to any of the measured meat quality or carcass traits as influenced by TSL. It is generally accepted that pigs with higher leaner growth efficiency also exhibit tougher meat [Similar to drip loss, the cooking loss measured after cooking the sample for shear force measurement, was influenced by MC (is trait . These rher meat . It shouher meat  and therp = 0.009)) that in TSL C, EM produced meat with a more pronounced red colour than entire males (line B) or immunocastrates (line B). Similar results were reported by Gispert et al. (2010) [Pigs originating from the TSL B had meat with a more pronounced red colour (CIE a*) compared to pigs originating from the TSL C . Althoug. (2010)  in (Land. (2010)  observed. (2010)  found noThe mean concentrations of androstenone and skatole in backfat between TSL and different MC, as well as the significance of both factors and their interactions, are shown in p < 0.001), while the concentration of skatole was influenced only by the MC (p = 0.0039). With regard to the TSL, the highest backfat mean androstenone level was demonstrated in EM and IC pigs from the TSL C (in average 1.51 µg per g of fat), while pigs from other two sire lines had similar and significantly lower androstenone levels , regardless of the TSL. It is known that marked differences in skatole levels in plasma and fat may exist between breeds  and highWith regard to MC, as expected, EM had significantly higher levels of androstenone (1.66 µg/g) and skatole (0.092 µg/g) in back fat compared to IC . The present results are consistent with the previous research studies that showed an effective reduction of compounds responsible for boar taint after effective immunocastration ,49,84.The proportion of the tainted samples   is preseOn the other hand, 79% of the entire males originating from TSL C, 40% of the pigs sired by TSL B boar and 35% of EM originating from TSL A exhibited androstenone levels above 1.00 µg/g. Despite the high incidence of elevated androstenone level, in TSL B and C, only 5% of the animals  had skatole concentration above 0.25 µg/g, and in EM originating from TSL A, skatole levels above the designated threshold were observed in 10% of the animals. It should be noted, however, that in the majority of animals with elevated androstenone/skatole levels, their values were close to the threshold.A significant interaction between MC and TSL existed for the androstenone level . EM fromThe results of the olfactory analysis together with confidence intervals for incidence of boar taint in EM and IC samples from three TSL are presented in Additionally, the disagreement between the sensory scores of the two different olfactory methods was also observed . For insInterestingly, although most of the tainted samples identified by the sensory panel originated from the TSL B, samples originating from the TSL C were those with the highest concentration of both boar taint compounds as determined by chemical analysis (79% vs. 35% for line A and 40% for line B). Unlike in samples from TSL A and B, with elevated levels of androstenone together with skatole concentration above 0.1 μg/g (data not shown), those samples had high androstenone levels, but their skatole levels were all lower than 0.1 μg/g fat. The research on the tainted patties showed that androstenone levels up to 2.07 μg/g in back fat are acceptable if skatole levels are low (≤0.10 μg/g), while skatole levels in back fat higher than 0.10 μg/g are not acceptable at any androstenone level . Low skaThe results of this research showed that interaction between TSL and MC plays a significant role in carcass and meat quality traits in modern fatteners and therefore should be taken into consideration when deciding on the production of a specific male category. The immunocastration proved to be most beneficial for the fatteners originating from TSL C, with a positive influence on carcass traits and without significant deterioration of meat quality. Terminal sire line B (regardless of the MC) produced fatteners with higher lean meat percentage and lower drip loss and WSBF values, with a more pronounced red colour of the meat. The EM from this TSL had longer carcasses and higher ham circumference, with similar meat quality traits compared to EM from the other two TSL. On the other hand, due to somewhat higher skatole levels (>0.1 μg/g), more IC and EM carcasses were scored as tainted, due to which precaution measures for avoiding boar taint should be implemented. As for the SC, the best performance in carcass traits was observed for animals sired by TSL B sire; however, SC from the TSL C exhibited to some extent better meat quality.Sensory analysis revealed the discrepancy between the used methods, with the microwave method classifying more samples into the tainted group. Due to observed discrepancy and the lack of threshold values in sensory evaluation, classifying the carcasses according to objective measurements of androstenone and skatole concentrations is still recommended.


Medicago sativa L.) is an important and widely cultivated forage grass. The productivity and forage quality of alfalfa are severely affected by salt stress. Melatonin is a bioactive molecule with versatile physiological functions and plays important roles in response to various biotic and abiotic stresses. Melatonin has been proven efficient in improving alfalfa drought and waterlogging tolerance in recent studies. In our reports, we applied melatonin exogenously to explore the effects of melatonin on alfalfa growth and salt resistance. The results demonstrated that melatonin application promoted alfalfa seed germination and seedling growth, and reduced oxidative damage under salt stress. Further application research found that melatonin alleviated salt injury in alfalfa plants under salt stress. The electrolyte leakage, malondialdehyde (MDA) content and H2O2 content were significantly reduced, and the activities of catalase (CAT), peroxidase (POD), and Cu/Zn superoxide dismutase (Cu/Zn-SOD) were increased with melatonin pretreatment compared to control plants under salt stress with the upregulation of genes related to melatonin and antioxidant enzymes biosynthesis. Melatonin was also involved in reducing Na+ accumulation in alfalfa plants. Our study indicates that melatonin plays a primary role as an antioxidant in scavenging H2O2 and enhancing activities of antioxidant enzymes to improve the salt tolerance of alfalfa plants.Alfalfa ( Medicago sativa L.) is an excellent perennial leguminous grass and one of the most important forage crops which is widely cultivated around the world. For its high protein content, rich nutrition value and high biomass yield, it is known as the “King of Forages”. Approximately 32.2 million hectares of alfalfa is planted worldwide, and with the development of livestock industries and new policies in China, alfalfa has become the most important and widely used forage grass in integrated farming systems in China [Alfalfa  is a bioactive molecule that was first identified in the mammalian pineal gland. It is a derivate from the essential amino acid tryptophan. Melatonin plays an important role in regulating circadian rhythms, sleep disorders, immunologic enhancement and antioxidative activity in animals ,22. MelaCu/Zn-SOD and Mn-SOD genes [M. hupehensis by directly scavenging H2O2 and enhancing the activities of antioxidative enzymes, and by controlling the expression of ion-channel genes to maintain homeostasis [Evidence shows that melatonin is a powerful antioxidant in plant responses to abiotic stresses ,33. UndeOD genes . ExogenoOD genes . Pretreaeostasis . Melatoneostasis ,48. BaseTo validate the hypothesis, we assessed the effects of various concentrations of melatonin on the germination and seedling growth of alfalfa under salinity conditions. The results showed that the exogenous application of melatonin can remarkably enhance salt tolerance and alleviate the salt injury of alfalfa. Our study confirms that melatonin performs its primary function as an antioxidant, positively improving the salt tolerance of alfalfa through scavenging reactive oxygen and enhancing the activities of antioxidant enzymes.Melatonin was proved beneficial to plant growth and development, as well as response to various abiotic stresses. Before salinity treatment, an appropriate salt and melatonin (MT) concentration was screened. The germination rate of alfalfa seeds under 200 mM and 250 mM NaCl was about 40% and 23%, respectively A,C. The 2O2 content especially with 50 µM MT pretreatment  represents the plant cell permeability, which is an important index of a plant’s salt tolerance . Malondireatment A–C. Despreatment D–G. To sTo assess the effects of melatonin on alfalfa plants, we applied 50 µM MT to one-month-old plants via foliar spraying for seven days. Half of the MT-pretreated plants and half of the control plants were subjected to salinity treatment with 200 mM NaCl. Fifteen days later, melatonin-treated plants under normal conditions were robust, which was similar to the control plants. Plants under salt stress showed retarded growth and with visible foliar injury, and a majority of leaves were wilted. However, melatonin-treated plants exposed to salt stress for 15 days exhibited obvious mitigating effects A,B.2O2 content was also increased in all four group plants after 15 days, while that in all plants with MT pretreatment was significantly lower than that in control plants under normal or salinity conditions  is an important peroxidase that widely exists in plants, and that belongs to the non-enzymatic system. It catalyzes glutathione (GSH) into glutathione disulfide (GSSG), causes poisonous peroxide reduction into non-toxic hydroxyl compounds and participates in H2O2 decomposition [Cu/Zn-SOD, CAT and APX genes in plants with MT pretreatment were upregulated under normal and salt stress conditions, which contributed to H2O2 scavenging to enhance the antioxidative ability of plants. However, the main function of melatonin on enzymatic or non-enzymatic system-mediated ROS scavenging in alfalfa plants still requires further study. The positive effects of melatonin on antioxidant enzymes system under salt stress conditions is also confirmed in tomato seedlings and maize seedlings [TDC, SNAT and ASMT genes were all upregulated with MT pretreatment under salt stress, which was dedicated to the biosynthesis of endogenous melatonin in plants. The relative expression level of ASMT was not increased significantly, which might have resulted from the alternative function of caffeic acid O-methyltransferase (COMT) in the last step of melatonin biosynthesis [The enhancement of salt tolerance through melatonin application mainly depends on directly scavenging ROS and enhancing the activities of antioxidative enzymes and photosynthetic efficiency, as well as regulating the transcription of genes related to salt stress . Melatonthan CAT ,54. In oposition ,56. The eedlings ,58. Exogynthesis .SNAT and ASMT, was an efficient way to modulate endogenous melatonin and to further reveal the melatonin regulation pathway in response to various stress conditions, especially with the discovery of the first melatonin receptor CAND2/PMTR1 [SNAT and ASMT genes from alfalfa and overexpressed them respectively into the alfalfa genome, and the elevated melatonin content in transgenic plants promoted transgenic plant growth. In the future, we would like to evaluate the abiotic stress resistance and explore the molecular mechanism of melatonin regulation with respect to plant resistance to abiotic stresses.The current studies were mainly dedicated to the exogenous application of melatonin so as to enhance various abiotic and biotic stresses. The effects of exogenous melatonin were discrepant with differences in treatment time, treatment organs, melatonin concentration and plant species. With the identification of genes in the melatonin biosynthesis pathway, to uncover the regulation mechanisms of melatonin in plants, the manipulation of genes expression in the melatonin biosynthesis pathway, mainly D2/PMTR1 . Based oIn summary, we evaluated the effects of exogenous melatonin on alfalfa germination, seedling growth, and plant resistance to salt stress. The results demonstrate that exogenous application of melatonin can promote plant growth and alleviate the salt damage of alfalfa plants under salt stress by upregulating the expression of genes related to melatonin biosynthesis and antioxidative enzyme activities. The salt tolerance of alfalfa was improved by reducing oxidative damage and enhancing the activities of antioxidant enzymes, which provides insight into how alfalfa salt tolerance can be improved with a novel bioactive molecule, melatonin.Medicago sativa L.) cultivar used in this experiment was “Zhongmu No. 2”. All chemicals used in experiments were of analytical grade. Melatonin (N-acetyl-5-methoxytryptamine) was purchased from Sigma-Aldrich  and stored at −20 °C.All of the experiments were conducted at China Agricultural University, Beijing . The alfalfa , germinated at 25 °C in darkness for 2 days, and then transferred to light conditions at 25/22 °C under a light/dark photoperiod of 16/8 h. One week later, the germination rates and the lengths of the seedling roots were measured. Three biological replicates were performed.To assess the melatonin effect upon salt stress, sterile seeds were immersed into melatonin solutions of various concentrations  for 24 h at 4 °C in the dark. After being air-dried, one hundred primed seeds were placed on 12 × 12 cm Petri dishes with three layers of filter paper moistened with 10 mL of distilled water or a 200 mM NaCl solution, germinated at 25 °C in the dark for 2 days, and then transferred to light conditions at 25/22 °C under a light/dark photoperiod of 16/8 h. Three replications of 100 seeds per plate were performed. The germination potential, germination rate, root length, fresh weight and root/shoot ratio of the plants with various concentrations of MT pretreatment were also recorded.−2s−2 light intensity at 25 ± 2 °C. Seedlings were watered with 1/2 × Hoagland solution every two days. One month later, when the height of the seedlings was about 15 cm, seedlings were divided into four groups with two control groups and two experiment groups. Control Group 2 (CK2) and the Experiment Group 2 (E2) were applied with 50 mL of a 50 µM melatonin solution by foliar spraying every night, while Control Group 1 (CK1) and Experiment Group 1 (E1) were sprayed with 50 mL of deionized water every night for one week. Hereafter, CK1 and CK2 were watered with a nutrient solution every other day, and E1 and E2 were watered with a nutrient solution with 250 mM NaCl every other day for another two weeks. Each group contained 12 pots with three plants per pot.Seedlings germinated on the filter paper with distilled water were transferred to plastic pots (15 cm in diameter) with a mixture of soil (vermiculite/humus = 1:1) and maintained in a growth room under a 16/8 h (light/dark) photoperiod with 200 µmol mElectrolyte leakage was determined according to our previous reports . The freMalondialdehyde (MDA) content was extracted and measured using the MDA Kits  according to the manufacturer’s instructions.The leaves of plants before and after salt stress treatment were sampled for catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) measurement with the corresponding plant kits  following the manufacturer’s instructions with a spectrophotometer . + and K+ content was measured according to our previous work [+ and K+ content. Three biological replicates were performed.The Naous work . The shohttp://plantgrn.noble.org/AGED/index.jsp). The relative expression levels of target genes were calculated with the formula 2−ΔΔCT method. All of the primers used in qRT-PCR are listed in Total RNA was isolated from leaves using an RNA Extraction Kit . The integrity of the RNA was ensured by gel electrophoresis and an absorbance measurement at 260 and 280 nm with a Nanodrop 2000. The intact RNA was reverse-transcribed with the PrimeScript RT reagent Kit with gDNA Eraser  according to the manufacturer’s instructions. Synthesized cDNA was subjected to PCR with an actin gene primer from alfalfa to determinate the quality [Each experiment was repeated three times and with at least three biological replications. All data were subjected to one-way analysis of variance , and multiple comparisons of the mean value were made via Duncan’s test.