Although massive cell atlases are already available, it is still a challenge to obtain an atlas of all tissues within a single body, especially in fetuses and pregnant mothers. We present a transcriptomic atlas of 2.56 million single cells covering 115 and 119 tissues from one fetal pig and its pregnant mother, respectively. We found that a cluster of heart capillary endothelial cells with enhanced fatty acid transit capability was enriched for pregnancy but restored after gestation. We also deciphered that l-leucine transport insufficiency in the trophoblast causes fetal growth restriction by reducing a muscle type II myofiber subcluster. Our "all-from-one" strategy enabled the identification of tissue cell type-specific transcription factors and provided insights into pregnancy heart adaptation and fetal growth restriction.

This study examines the S2 protein of the Nephropathogenic Infectious Bronchitis virus (NIBV), essential for the virus's adaptation and expanded tissue and host antigenic epitopes, secondary and tertiary structures were analyzed. The NIBV-S2 gene was amplified via RT-PCR, cloned into the pET-32a (+) vector to form the recombinant plasmid pET-SX9-S2, and transferred into BL21(DE3) cells. An antirHis-S2 polyclonal antibody was generated using the expressed rHis-S2 as the antigen. The indirect ELISA titer of this polyclonal antibody was 1:1024,000. This polyclonal antibody against NIBV-S2 effectively detects NIBV-S2 and is suitable for western bloting, immunofluorescence, and Co-IP experiments. Detection showed a marked rise in NIBV-S2 protein expression from 3 to 9 dpi, peaking at 9 dpi, followed by a decline from 9 to 15 dpi as the infection progressed. At 9 dpi, there was a notable downregulation of the ABCG2, a uric acid transporter, in the kidneys of NIBV-infected chickens compared to the control group. Furthermore, immunoprecipitation (Co-IP), and molecular dynamics (MD) simulations have demonstrated that the NIBV-S2 protein possesses the ability to bind to ABCG2. This observation was further substantiated by in vitro inhibition experiments, which revealed that the expression level of the S protein decreased following the application of the ABCG2 inhibitor Ko143. In summary, NIBV utilizes its S2 protein to interact with ABCG2, thereby facilitating viral colonization in the kidneys, which may contribute to urate accumulation.

While previous studies have extensively demonstrated that summer heat stress (HS) impairs oocyte quality via follicular granulosa cell (GC) mediation, the molecular mechanisms underlying HS-induced GC-mediated oocyte damage—particularly at the multi-omics level—remain poorly understood. This study integrated metabolomic and transcriptomic analyses of GCs from gilts under seasonal thermal stress (winter CON vs. summer HS) to elucidate GC-mediated regulatory networks affecting oocyte quality. Non-targeted metabolomics identified 45 differentially accumulated metabolites (DAMs, p < 0.05), with 69% being lipids/lipid-like molecules enriched in pathways such as glycerophospholipid metabolism, choline metabolism, linoleic acid metabolism, the adipocytokine signaling pathway, and the sphingolipid signaling pathway. Parallel transcriptomics revealed 9085 differentially expressed genes (DEGs, Padj < 0.05), of which the predominant genes were associated with lipid metabolism, hormone synthesis, and cellular senescence pathways. Cross-omics integration highlighted significant correlations between DAMs and DEGs, particularly for lysoPC(20:4) and 1-hexadecyl-2-eicosatrienoyl-sn-glycero-3-phosphocholine, which showed co-regulation with 69 and 48 genes, respectively. Notably, candidate genes like TMEM94, SLIT3, DACT3, and CEBPD, were identified as key regulators of GCs metabolic reprogramming. This study demonstrates for the first time that in vivo HS compromises oocyte developmental competence by disrupting the GC metabolic activities, particularly through lipid metabolism and associated pathways. The identified metabolic signatures and regulatory genes offer mechanistic insights into seasonal infertility and potential biomarkers for thermo-protective strategies in swine reproduction.

The pivotal roles of post-transcriptional N6-methyladenosine (m6A) modification in animal muscle development regulation have been wildly reported previously. However, whether this epigenetic mechanism also takes part in the muscle fiber differentiation, which remarkedly affects the meat quality and production in livestock, remains unknown. In this study, an eight-breeds crossbreed mosaic pig population consisted by 302 adult pigs was constructed to estimate the myofiber phenotypes. We found that despite the same genetic background, remarkable differences of longissimus dorsi muscle fiber types still existed between different subsets of this population. m6A abundance was evaluated subsequently in the subset samples with extremely low (Low group) or high (High group) proportions of type Ⅱb myofibers based on methylated RNA immunoprecipitation sequencing (MeRIP-seq) analysis. A total of 27,699 and 28,194 high-confidence m6A peaks were identified in Low and High group samples, respectively. Among them, 8128 differential peaks shared between two groups were screened out and were annotated into 189 differentially expressed genes (DEGs). Gene ontology analysis revealed that 22 of DEGs such as MYH7, TPM3, TNNT1, FHL1, CAMTA2 and MYL3, were involved in the skeletal muscle development regulations. Furthermore, 21 of these candidate genes were validated to regulate the myofiber differentiation in the mosaic pig population through quantitative trait transcript analysis. The findings of this study demonstrated the critical roles of m6A modification in porcine longissimus dorsi muscle fiber differentiation regulation, and provide us with new clues in improving the animal growth rate and meat quality during the future husbandry production.

Objective: Animals will experience energy deprivation processes such as moulting, clutching, migration and long-distance transportation under natural survival conditions and in production practices, and the body will trigger a series of adaptive metabolic changes during these processes. Fasting and refeeding after fasting can induce remodeling of nutrients and energy metabolism. This study aims to investigate the mechanisms by which the gut microbiota and liver of poultry respond to energy deprivation under specific conditions.Methods: Ninety 252-day-old laying hens were randomly divided into 3 groups: (1) fed ad libitum (control group); (2) fasted from day 13 to day 17 (fasting group); (3) fasted from day 1 to day 5, then refed on a specific feeding way (refeeding group). After that, the serum, liver, jejunum tissues, and cecum contents were sampled and sent for metabolome, transcriptome, morphology, and 16S rDNA sequencing analyses, respectively.Results: Results showed that food deprivation not only observably decreased the body weight, liver index, and the villus height and villus/crypt ratio of jejunum, but also significantly changed the gut microbiota compositions, serum metabolic profiles, and the hepatic gene expression patterns of laying hens, whereas these changes were effectively reversed by the following refeeding operation. At the same time, metabolome combined transcriptome analysis revealed that both serum differential metabolites and hepatic differential expressed genes (DEGs) were consistently enriched in the lipid and amino metabolism pathways, and strong correlations were synchronously found between the differential metabolites and both of the differential gut microbial genera and DEGs, suggesting the crosstalks among gut, liver and their resulting serum metabolic products.Conclusion: The results suggested that the organism might coordinate to maintain metabolic homeostasis under energy deprivation through a combination of changes in gut microbial composition and hepatic gene expression.

Most of follicles undergo a degenerative process called follicular atresia. This process directly affects the egg production of laying hens and is regulated by external and internal factors. External factors primarily include nutrition and environmental factors. In follicular atresia, internal factors are predominantly regulated at 3 levels; organic, cellular and molecular levels. At the organic level, the hypothalamic-pituitary-ovary (HPO) axis plays an essential role in controlling follicular development. At the cellular level, gonadotropins and cytokines, as well as estrogens, bind to their receptors and activate different signaling pathways, thereby suppressing follicular atresia. By contrast, oxidative stress induces follicular atresia by increasing ROS levels. At the molecular level, granulosa cell (GC) apoptosis is not the only factor triggering follicular atresia. Autophagy is also known to give rise to atresia. Epigenetics also plays a pivotal role in regulating gene expression in processes that seem to be related to follicular atresia, such as apoptosis, autophagy, proliferation, and steroidogenesis. Among these processes, the miRNA regulation mechanism is well-studied. The current review focuses on factors that regulate follicular atresia at organic, cellular and molecular levels and evaluates the interaction network among these levels. Additionally, this review summarizes atretic follicle characteristics, in vitro modeling methods, and factors preventing follicular atresia in laying hens.

BACKGROUND:Fatty liver hemorrhagic syndrome (FLHS) in the modern poultry industry is primarily caused by nutrition. Despite encouraging progress on FLHS, the mechanism through which nutrition influences susceptibility to FLHS is still lacking in terms of epigenetics.RESULTS:In this study, we analyzed the genome-wide patterns of trimethylated lysine residue 27 of histone H3 (H3K27me3) enrichment by chromatin immunoprecipitation-sequencing (ChIP-seq), and examined its association with transcriptomes in healthy and FLHS hens. The study results indicated that H3K27me3 levels were increased in the FLHS hens on a genome-wide scale. Additionally, H3K27me3 was found to occupy the entire gene and the distant intergenic region, which may function as silencer-like regulatory elements. The analysis of transcription factor (TF) motifs in hypermethylated peaks has demonstrated that 23 TFs are involved in the regulation of liver metabolism and development. Transcriptomic analysis indicated that differentially expressed genes (DEGs) were enriched in fatty acid metabolism, amino acid, and carbohydrate metabolism. The hub gene identified from PPI network is fatty acid synthase (FASN). Combined ChIP-seq and transcriptome analysis revealed that the increased H3K27me3 and down-regulated genes have significant enrichment in the ECM-receptor interaction, tight junction, cell adhesion molecules, adherens junction, and TGF-beta signaling pathways.CONCLUSIONS:Overall, the trimethylation modification of H3K27 has been shown to have significant regulatory function in FLHS, mediating the expression of crucial genes associated with the ECM-receptor interaction pathway. This highlights the epigenetic mechanisms of H3K27me3 and provides insights into exploring core regulatory targets and nutritional regulation strategies in FLHS.

The adverse effects of chronic heat stress (CHS)-induced fatty liver syndrome on laying hens during the egg-producing stages have been wildly documented. However, until nowadays, the CHS responses of growing laying hens as well as its alleviating effects of vitamin C are rarely reported. In this study, 12-wk-old laying hens were subjected to CHS at 36 degrees C for 10 h/d for 3 wk with or without dietary supplementation of 300 mg/kg vitamin C. Results showed that CHS significantly impaired the growth performances and the liver functions of birds, as characterized by reduced feed intake and body weight, increased hepatic lipid accumulation and serum concentrations of TG, ALT, and AST, as well as the abnormal expression patterns of the lipid metabolism-related genes. Vitamin C supplementation successfully mitigated the lipid accumulation, while showing no alleviating effect on the serum contents of ALT or AST, which are two key indicators of liver functions. Metabolomic analysis based on UPLC-Q-TOF/MS identified 173 differential metabolites from the HS and HSV group samples, and they are mainly enriched in the pathways related to the cellular components, vitamin and amino acid metabolism and energy substance metabolism. The results indicate that CHS-induced hepatic lipid deposition in growing laying hens is effectively alleviated by dietary supplementation of vitamin C, which is probably resulted from the alterations of hepatocellular metabolic patterns.

Epigenetic regulation of gene expression has been reported in the pathogenesis of metabolic disorders such as diabetes and liver steatosis in humans. However, the molecular mechanisms of fatty liver hemorrhagic syndrome (FLHS) in chickens have been rarely studied. H3K27ac chromatin immunoprecipitation coupled with high-throughput sequencing and high-throughput RNA sequencing was performed to compare genome-wide H3K27ac profiles and transcriptomes of liver tissue between healthy and FLHS chickens. In total, 1,321 differential H3K27ac regions and 443 differentially expressed genes were identified (| log2Fold change| ≥ 1 and P -value ≤ 0.05) between the two groups. Binding motifs for transcription factors involved in immune processes and metabolic homeostasis were enriched among those differential H3K27ac regions. Differential H3K27ac peaks were associated with multiple known FLHS risk genes, involved in lipid and energy metabolism ( PCK1 , APOA1 , ANGPTL4 , and FABP1 ) and the immune system ( FGF7 , PDGFRA , and KIT ). Previous studies and our current results suggested that the high-energy, low-protein (HELP) diet might have an impact on histone modification and chromatin structure, leading to the dysregulation of candidate genes and the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which causes excessive accumulation of fat in the liver tissue and induces the development of FLHS. These findings highlight that epigenetic modifications contribute to the regulation of gene expression and play a central regulatory role in FLHS. The PPAR signaling pathway and other genes implicated in FLHS are of great importance for the development of novel and specific therapies for FLHS-susceptible commercial laying hens.

On modern farms, animals are at high risk of bacterial invasion due to environmental stress factors. The adrenal gland is the terminal organ of the stress response. The crosstalk between adrenal endocrine stress and innate immune response is critical for the maintenance of immune homeostasis during inflammation. Thus, it's important to explore whether stresses play a pivotal role in lipopolysaccharide (LPS)-induced inflammatory response in the porcine adrenal gland. Thirty-days-old Duroc x Landrace x Large White crossbred piglets (12 +/- 0.5 kg) were randomly allocated into four groups in a 2 x 2 factorial arrangement of treatments, including ACTH pretreatment (with or without ACTH injection) and LPS challenge (with or without LPS injection). Each group consisted of six male piglets. The results showed that our LPS preparation alone induced mRNA expressions of IL-1 beta, IL-6, TNF-alpha, IL-10, COX-2, TLR2, TLR4, and GR (P< 0.05). ACTH pretreatment downregulated the TLR2 mRNA and IL-6 protein level induced by our LPS preparation significantly (P< 0.05) by one-way ANOVA analysis. Treatment with LPS alone extremely significantly decreased ssc-miR-338 levels (P< 0.01). Interaction of ACTH x LPS was significant for cNOS level (P= 0.011) and ssc-miR-338 expression (P= 0.04) by two-way ANOVA analysis. The LPS treatment significantly downregulated cNOS levels (P< 0.01), which was significantly attenuated by ACTH pretreatment (P< 0.05). Lipopolysaccharide alone did not affect ssc-miR-146b expression levels compared to that in the vehicle group. However, ACTH pretreatment in combination with LPS significantly increased this micro-RNA expression (P< 0.05). TLRs 1-10 were all expressed in adrenal tissue. The LPS challenge alone induced remarkable compensatory mitochondrial damages at the ultrastructural level, which was alleviated by ACTH pretreatment. Accordingly, ACTH pretreatment was able to block LPS-induced secretion of local adrenal cortisol (P< 0.05). Taken together, our results demonstrate that ACTH pretreatment seems to attenuate LPS-induced mitochondria damage and inflammation that decreased cNOS activity in the adrenal gland and ultimately returned local adrenal cortisol to basal levels at 6 h post LPS injection.

The oocyte is vulnerable to various environmental stressors, including heat exposure. Cumulus-oocyte complexes (COCs) comprise functional units for oocytes in vitro maturation, and the cumulus cells provide essential supports and protect the oocyte from environmental insults. Heat exposure results in varied consequences in oocyte, presumably due to different responses of cumulus cells to heat exposure. In this study, we examined whether heat exposure of different duration affects porcine oocytes quality differently, and how such effects, if any, relate to transcriptomic profiles of cumulus cells. COCs were heat-exposed for 4 h (20-24 h, COC4) and 24 h (0-24 h, COC24), respectively, and the quality of oocytes in COC24 group showed significantly impaired with disrupted cumulus expansion and extracellular matrix (ECM) structure. The transcriptomic analysis identified 749 and 1238 differential expression genes (DEGs) in COC4 and COC24, respectively. Moreover, 852 DEGs were found when COC24 was compared with COC4, and the downregulated DEGs were mainly associated with Gene Ontology terms linked with ECM and cell proliferation. In the protein-protein interaction network, HSPE1, TNFAIP6, COL12A1, and COL18A1 were identified as hub genes playing important roles in heat-induced transcriptomic responses. These results indicate that impaired cumulus proliferation and ECM structure are responsible for heat-induced damage in oocytes quality.

Background Coccidiosis is caused by Eimeria spp. and can result in severe economic losses to the global poultry industry. Due to anticoccidial drug resistance rapidly developing in the parasites and drug residues in poultry products, efficacious and safe alternative coccidia control measures are needed. The objective of the present study was to identify common protective antigens which may be used as vaccine candidates in the development of subunit, multivalent, cross-protective vaccines against most of the economically important Eimeria species. Methods Whole sporozoite proteins of Eimeria acervulina were prepared and analyzed by 2-dimensional gel electrophoresis (2-DE) followed by western blotting using immune sera specific to E. tenella , E. acervulina , or E. necatrix . The protein spots detected by all three immune sera were then excised from the preparative gel and protein ID was performed by MALDI-TOF-MS/MS. Results Approximately 620 E. acervulina sporozoite protein spots were demonstrated by 2-DE with silver staining, among which 23 protein spots were recognized by immune sera specific to all three Eimeria species. The results showed that 21 putative E. acervulina proteins were identified, which include proteins with known enzymatic properties, and those which are involved in protein translation, transport and trafficking, and ribosomal biogenesis and functions. There is one protein which may be involved in transcription and one heat-shock protein. Two proteins contain predicted domains, but with no apparent functions known. There were 2 protein spots which had no detectable proteins. None of the proteins has a predicted signal peptide or a transmembrane domain; however, 6 of the 21 putative proteins were predicted to be potentially secretory through the non-classical pathway. Conclusions Our study identified a diverse group of antigens immunologically common to all three Eimeria species, none of which was previously characterized and tested as a vaccine candidate. Further research on immunogenicity and cross-protective potential of these individual proteins as vaccine candidates will aid the development of vaccines against the most common and pathogenic Eimeria spp.

Objectives: Glucocorticoid receptor (GR) expressed in hippocampus is critical for the homeostasis of stress responses and susceptible to epigenetic modulation caused by maternal factors. Here we show that maternal methyl nutrition causes sex-biased changes in hippocampal expression of GR exon 1 mRNA variants, associated with promoter DNA methylation, across two offspring generations in rats.Methods: Three-month-old female Sprague-Dawley rats (F0) were fed a diet supplemented with 1% betaine throughout the gestation and lactation. F0 dams and their F1 and F2 offspring of both sexes at weaning were used in the study.Results: A sex-specific transgenerational effect was observed. F2 females, but not males, followed the same pattern of their grand dams showing increased mRNA expression of total GR and its exons 1.4, 1.7, 1.10 and 1.11 variants coincided with promoter DNA hypomethylation in the hippocampus. However, F1 females, but not males, exhibited an opposite pattern, showing decreased expression of GR and its mRNA variants accompanied with promoter hypermethylation. The protein content of phospho-GR and BDNF/ERK in the hippocampus displayed the same sex and generation specificity.Discussion: These results indicate that maternal betaine exerts transgenerational effects on hippocampal GR expression and BDNF/ERK pathway in female rat offspring, with generation-dependent patterns of DNA methylation on alternative GR promoters.

This study evaluated the effect of maternal restraint stress during the gestation period on behaviors, biochemical parameters, and antioxidant capacities of offspring rats (Rattus norvegicus (Berkenhout,1769)) at weaning age. Behaviors, plasma biochemical indices, and antioxidant ability of the liver, soleus muscle, and gastrocnemius muscle of mother and (or) offspring rats were analyzed. Significant increases were found in the immobility and swinging behavior frequencies of offspring male rats; no difference was found in behaviors of female rats. The antioxidant indices including superoxide dismutase, nitric oxide synthase, and total antioxidant capacity in the soleus muscle of offspring male rats were significantly decreased in the restraint group. Female offspring rats showed significant lower glutathione and higher malondialdehyde levels in the gastrocnemius muscle and liver, respectively. No difference was found in the productive performance and plasma biochemical indices of maternal rats, nor in the biochemical parameters of the two groups of weaning rats. The results suggested that maternal chronic stresses negatively affected the behaviors and antioxidant abilities of offspring rats, and that these effects possibly have a greater impact on offspring male rats than on female rats.

Cumulus cells (CCs) play critical roles in oocytes maturation through intercellular communication. The adverse effect of heat stress (HS) on oocyte maturation has been well documented, whereas the HS responses of CCs and the oocytes in association with cumulusoocyte communication remain unclear. In this study, cumulus‐oocyte complexes (COCs) were subjected to HS at 42°C for 24 h during in vitro maturation. Cumulus expansion was impaired and oocyte quality was reduced with lower survival rate, polar body extrusion rate and early embryo developmental potentials. Oocytes and cumulus cells isolated from COCs demonstrated distinct responses to HS. In oocytes, mitochondrial DNA (mtDNA)‐encoded genes and heat shock protein (HSP)‐related genes were significantly down‐regulated, with significantly lower ATP content yet higher mitochondria reactive oxygen species (mROS) and caspase 3 activity. In contrast, CCs isolated from heat‐exposed COCs showed significant activation of mtDNAencoded genes and HSP‐related genes, which was accompanied by increased ATP content and caspase 3 activity yet unaltered mROS content. When denuded oocytes and isolated CCs were subjected to HS separately, ATP content in denuded oocytes decreased more extensively than that in oocytes isolated from heat‐exposed COCs. Also, mROS was significantly increased in CCs but not in denuded oocytes, which coincided with less pronounced increase of caspase 3 in denuded oocytes. Moreover, F‐actin, a marker of trans‐zonal projections, and occludin, a tight junction protein, and their co‐localization, were significantly reduced in heat‐exposed COCs. These results indicate that cumulus cells and oocytes show distinct HS responses, indicating disrupted cumulus‐oocytes communication in heat‐exposed porcine oocytes. Support or Funding Information This work was supported by the National Key Research and Development Program of China (2016YFD0500502), the National Basic Research Program of China (2014CB138502), the Priority Academic Program Development of Jiangsu Higher Education Institutions, and Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Homocysteine (Hcy) is an intermediate in the one-carbon metabolism that donates methyl groups for methylation processes involved in epigenetic gene regulation. Although poor oocyte quality in polycystic ovarian syndrome (PCOS) patients is associated with elevated Hcy concentration in serum and follicular fluid, whether Hcy directly affects oocyte quality and its mechanisms are poorly understood. Here we show that Hcy treatment impaired oocyte quality and developmental competence, indicated by significantly reduced survival rate, polar body extrusion rate, and cleavage rate. Hcy treatment resulted in mitochondrial dysfunction, with increased production of mitochondrial ROS, reduced mtDNA copy number, and the expression of 7 out of 13 mtDNA-encoded genes and 2 ribosome RNA genes, 12S rRNA and 16S rRNA. Upon Hcy treatment, the expression of one-carbon metabolic enzymes and DNMT1 was enhanced. Interestingly, DNA methyltransferase inhibitor 5AZA rescued Hcy-induced mitochondrial dysfunction, impaired oocyte quality and developmental competence. Concurrently, expression of one-carbon metabolic enzymes and methylation status of mtDNA coding sequences were also normalized, at least partially, by 5 ' AZA treatment. Our findings not only extend the understanding about how Hcy induces poor oocyte quality, but also contribute to a novel angle of identifying targets for enhancing the quality of oocyte from PCOS patients. Homocysteine disrupts one-carbon metabolism, which leads to hypermethylation of mitochondrial DNA, mitochondrial dysfunction, and impaired oocyte quality.

Cumulus cells (CCs), the granulosa cells surrounding the oocytes, play critical roles in oocytes maturation through intercellular communication by extending trans‐zonal projections (TZPs) to contact oocytes via gap junctions (GJs). The adverse effect of heat stress (HS) on oocyte maturation has been well documented, whereas the HS responses of CCs and the oocytes in association with GJ/TZP colocalization remain unclear. In this study, porcine cumulus‐oocyte complexes (COCs) were subjected to HS at 41.5°C for 24 hr during in vitro maturation. Cumulus expansion was impaired and oocyte quality was reduced with lower survival rate, polar body extrusion rate, and early embryo developmental potentials. CCs and oocytes isolated from COCs demonstrated distinct responses to HS. The messenger RNA abundance of heat shock protein‐related genes and mitochondrial DNA‐encoded genes, together with ATP content, were significantly increased in CCs, yet decreased in oocytes, despite activation of caspase 3 detected in both CCs and oocytes. Similar changes were observed when denuded oocytes and isolated CCs subjected to HS separately, except mitochondria reactive oxygen species (mROS). In heat‐stressed COCs, mROS was significantly increased only in oocytes. However, when isolated CCs and denuded oocytes were heat‐stressed separately, mROS was significantly increased only in CCs. Moreover, F‐actin, a TZP marker, and its colocalization with a GJ protein connexin‐45, were significantly reduced in heat‐exposed COCs. These results indicate that HS induces distinct responses in porcine CCs and oocytes in association with disrupted GJ and TZP colocalization.

Currently, the effects of stress on the body metabolism are attracting more and more attentions. To better understand the probable stress-induced metabolic changes, the changes in plasma indices and hepatic proteome were investigated in pigs fitted with jugular venous catheters administered adrenocorticotropic hormone (ACTH), intramuscularly twice daily for 7 days. In particular, plasma hormones, blood cells and biochemical indicators were analyzed. In addition, the hepatic proteome was analyzed by two-dimensional electrophoresis and matrix-assisted laser desorption ionization time of flight mass spectrometry. The results revealed that after the initial ACTH injection, plasma ACTH, cortisol, triiodothyronine and tumor necrosis factor α increased (P ˂ 0.05). Additionally, lactic dehydrogenase activity increased (P < 0.05), and both leukocyte and lymphocyte count decreased (P < 0.05). In comparison, after the ACTH injection for 7 days, hepatic proteomics analysis identified 27 significantly changed proteins and most of these proteins (49%) are involved in metabolism, but plasma indices showed no obvious change. In conclusion, compared with the obvious changes in plasma indices after the initial ACTH injection, the multiple ACTH injections did not change plasma indices, yet the hepatic proteome profile was significantly altered. The results provide useful information for a better understanding of the effects of stress on metabolism.

The objective of present study was to investigate changes in salivary components during restraint to identify potential markers of stress. Pigs were subjected to a nasal snare stress (Experiment 1) or an immobilization stress (Experiment 2) by being enclosed in a steel cage. Saliva was collected before, during and after the stress, respectively. Salivary cortisol, serum amyloid A (SAA), haptoglobin (HP), chromogranin A (CgA), amylase, K+, Ca2+ and lactoferrin content were detected. The results showed that in Experiment 1, HP and CgA content increased significantly at 10min during the restraint (P<0.05, P<0.05), in agreement with the significantly increased cortisol and SAA levels (P<0.01, P<0.05), while amylase, K+ and lactoferrin concentrations did not significantly change. In Experiment 2, salivary HP and CgA concentrations also changed significantly during the restraint (P<0.01, P<0.01), yet cortisol, SAA, amylase, K+ and lactoferrin levels did not show obvious change. The results confirmed that salivary HP and CgA content may be useful candidate biomarkers to monitor the physical state in pigs during stress.

Melatonin, the major pineal secretory product, has a significant impact on the female reproductive system. Recently, the beneficial effects of melatonin on mammalian oocyte maturation and embryonic development have drawn increased attention. However, the exact underlying mechanisms remain to be fully elucidated. This study demonstrates that supplementing melatonin to in vitro maturation (IVM) medium enhances IVM rate, lipid droplets (LDs) accumulation as well as triglyceride content in porcine oocytes. Decrease of mitochondrial membrane potential, mitochondrial respiratory chain complex IV activity as well as mitochondrial reactive oxygen species (mROS) content indicated that melatonin induced a decrease of mitochondrial activity. The copy number of mitochondrial DNA (mtDNA) which encodes essential subunits of oxidative phosphorylation (OXPHOS), was not affected by melatonin. However, the expression of mtDNA-encoded genes was significantly down-regulated after melatonin treatment. The DNA methyltransferase DNMT1, which regulates methylation and expression of mtDNA, was increased and translocated into the mitochondria in melatonin-treated oocytes. The inhibitory effect of melatonin on the expression of mtDNA was significantly prevented by simultaneous addition of DNMT1 inhibitor, which suggests that melatonin regulates the transcription of mtDNA through up-regulation of DNMT1 and mtDNA methylation. Increase of triglyceride contents after inhibition of OXPHOS indicated that mitochondrial quiescence is crucial for LDs accumulation in oocytes. Taken together, our results suggest that melatonin-induced reduction in mROS production and increase in IVM, and LDs accumulation in porcine oocytes is mediated by mitochondrial quiescence.