Transcriptomic and metabolomic dissection of skeletal muscle of crossbred Chongming white goats with different meat production performance.

BACKGROUND: The transcriptome and metabolome dissection of the skeletal muscle of high- and low- growing individuals from a crossbred population of the indigenous Chongming white goat and the Boer goat were performed to discover the potential functional differentially expressed genes (DEGs) and differential expression metabolites (DEMs). RESULTS: A total of 2812 DEGs were detected in 6 groups at three time stages (3,6,12 Month) in skeletal muscle using the RNA-seq method. A DEGs set containing seven muscle function related genes (TNNT1, TNNC1, TNNI1, MYBPC2, MYL2, MHY7, and CSRP3) was discovered, and their expression tended to increase as goat muscle development progressed. Seven DEGs (TNNT1, FABP3, TPM3, DES, PPP1R27, RCAN1, LMOD2) in the skeletal muscle of goats in the fast-growing and slow-growing groups was verified their expression difference by reverse transcription-quantitative polymerase chain reaction. Further, through the Liquid chromatography-mass spectrometry (LC-MS) approach, a total of 183 DEMs in various groups of the muscle samples and these DEMs such as Queuine and Keto-PGF1α, which demonstrated different abundance between the goat fast-growing group and slow-growing group. Through weighted correlation network analysis (WGCNA), the study correlated the DEGs with the DEMs and identified 4 DEGs modules associated with 18 metabolites. CONCLUSION: This study benefits to dissection candidate genes and regulatory networks related to goat meat production performance, and the joint analysis of transcriptomic and metabolomic data provided insights into the study of goat muscle development.

Mitophagy is involved in the mitochondrial dysfunction of vitrified porcine oocytes.

Mitochondrial dysfunction is considered a crucial factor aggravating oocyte viability after vitrification-warming. To clarify the role of mitophagy in mitochondrial extinction of vitrified porcine oocytes, mitochondrial function, ultrastructural characteristics, mitochondria-lysosomes colocalization, and mitophagic proteins were detected with or without chloroquine (CQ) treatment. The results showed that vitrification caused mitochondrial dysfunction, including increasing reactive oxygen species production, decreasing mitochondrial membrane potential, and mitochondrial DNA copy number. Damaged mitochondrial cristae and mitophagosomes were observed in vitrified oocytes. A highly fused fluorescence distribution of mitochondria and lysosomes was also observed. In the detection of mitophagic flux, mitophagy was demonstrated as increasing fluorescence aggregation of microtubule-associated protein light chain 3B (LC3B), enhanced colocalization between LC3B, and voltage-dependent anion channels 1 (VDAC1), and upregulated LC3B-II/I protein expression ratio. CQ inhibited the degradation of mitophagosomes in vitrified oocytes, manifested as decreased mitochondria-lysosomes colocalization, increased fluorescence fraction of VDAC1 overlapping LC3B, increased LC3B-II/I protein expression ratio, and p62 accumulation. The inhibition of mitophagosomes degradation by CQ aggravated mitochondrial dysfunction, including increased oxidative damage, reduced mitochondrial function, and further led to loss of oocyte viability and developmental potentiality. In conclusion, mitophagy is involved in the regulation of mitochondrial function during porcine oocyte vitrification.

Addition of butylated hydroxytoluene (BHT) in tris-based extender improves post-thaw quality and motion dynamics of dog spermatozoa.

The aim of this study was to evaluate the interaction of different concentrations of butylated hydroxytoluene (BHT) in a tris-based extender on semen quality parameters in post-thawed dog semen. Twenty-four ejaculates were collected from eight male Beagle dogs using an artificial vagina. Pooled semen was diluted with a tris-based extender supplemented with 0 (control), 0.5, 1.0, 1.5, and 2.0 mM BHT, at a final concentration of 200 × 106 spermatozoa/mL. After thawing, sperm samples were assessed for motility parameters (CASA), membrane integrity (SYBR-14/PI), acrosome integrity (FITC-PNA), mitochondrial activity (JC-1/PI), malondialdehyde (MDA) concentration, and glutathione peroxidase (GPx) activity. The total motility, progressive motility, and average path velocity of the frozen-thawed sperm were significantly higher in the BHT1.5 group than in the control and the other sample groups (P < 0.05). Higher values of straight-line velocity, curvilinear velocity, amplitude of the lateral head displacement, and linearity were observed in the BHT1.0, BHT1.5, and BHT2.0 groups than in the control (P < 0.05). The BHT1.0 and BHT1.5 groups had higher percentages of straightness and acrosome integrity than the other groups (P < 0.05). Beat cross frequency, plasma membrane integrity, and GPx activity of the BHT1.5 and BHT2.0 groups were higher than those of the control (P < 0.05). A lower concentration of MDA was observed in the BHT1.0, BHT1.5, and BHT2.0 groups than in the control (BHT0) (P < 0.05). Our results indicate that 1.5 mM BHT is the optimal concentration for improving the post-thaw quality of canine spermatozoa.

Effect of substituting different concentrations of soybean lecithin and egg yolk in tris-based extender on goat semen cryopreservation.

This study aimed to investigate the effects of different concentrations of soybean lecithin (SL; 0.5%, 1%, and 1.5%) and egg yolk (EY) in Tris-based extenders on the semen quality parameters of post-thawed goat semen. Sixteen ejaculates were collected from eight healthy, mature Chongming White goats (3-5 years of age). Each ejaculate was divided into five equal aliquots, and then each pellet was diluted with one of the five Tris-based extenders containing 20% EY, 0.5% SL, 1% SL, 2% SL, or 3% SL. The cooled diluted semen was loaded into 0.5 mL polyvinyl French straws and cryopreserved in liquid nitrogen. Frozen semen samples were thawed at 37 °C and assessed for sperm motility, viability, plasma acrosome integrity, membrane integrity, and mitochondria integrity, and the spermatozoa were assessed for reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA). The semen extended in the 2.0% SL extract tended to have a higher sperm viability (57.44%), motility (52.14%), membrane integrity (45.31%), acrosome integrity (52.96%), and mitochondrial activity (50.21%) than the other SL-based extender concentrations (P < 0.05). The 2.0% SL treatment group was equivalent to the semen extended in 20% EY (P > 0.05). The extenders supplemented 20% EY or 2.0% SL significantly increased the SOD activity and decreased the ROS and MDA activities compared to the other groups (P < 0.05). In conclusion, the extenders supplemented with 20% EY and 2.0% SL had similar effects on spermatozoa preservation. These results indicate that a soybean lecithin-based diluent may be used as an alternative extender to egg yolk for the cryopreservation of goat semen.

Optimizing treatment of DNA methyltransferase inhibitor RG108 on porcine fibroblasts for somatic cell nuclear transfer.

Aberration in DNA methylation is believed to be one of the major causes of abnormal gene expression and inefficiency of somatic cell nuclear transfer (SCNT). RG108, a non-nucleoside DNA methyltransferase (DNMT) inhibitor, has been reported to facilitate somatic nuclear reprogramming and improved blastocyst formation. The aim of this study was to investigate interaction effect of RG108 treatment time (24-72 hr) and concentrations (0.05-50 µM) on donor cells, and further to optimize the treatment for porcine SCNT. Our results showed that RG108 treatment resulted in time-dependent decrease of genome-wide DNA methylation on foetal fibroblasts, which only happened after 72-hr treatment in our experiments, and no interaction effect between treatment time and concentration. Remarkable decrease of methylation in imprinted gene H19 and increased apoptosis was observed in 5 and 50 µM RG108-treated cells. Furthermore, the blastocyst rates of SCNT embryos were increased as the fibroblasts treated with RG108 at 5 and 50 µM, and additional treatment during cultivation of SCNT embryos would not provide any advantage for blastocyst formation. In conclusion, the RG108 treatment of 72 hr and 5 µM would be optimized time and concentration for porcine foetal fibroblasts to improve the SCNT embryonic development. In addition, combined treatment of RG108 on donor cells and SCNT embryos would not be beneficial for embryonic development.

Dietary rumen-protected arginine and N-carbamylglutamate supplementation enhances fetal growth in underfed ewes.

The present study was conducted with an ovine intrauterine growth restriction (IUGR) model to test the hypothesis that dietary rumen-protected l-arginine (RP-Arg) or N-carbamylglutamate (NCG) supplementation in underfed ewes is effective in enhancing fetal growth. Between Days 35 and 110 of pregnancy, 32 multiparous ewes carrying two fetuses were randomly assigned to one of four groups: a control (CG) group (n=8; 100% National Research Council (NRC) requirements for pregnant sheep), a nutrient-restricted (RG) group (n=8; fed 50% NRC requirements, and two treatment (ARG and NCG) groups (n=8 in each group; fed 50% NRC requirements supplemented with 20gday-1 RP-Arg or 5gday-1 NCG. All ewes were killed on Day 110 of pregnancy to determine fetal weight and fetal organ weights, and metabolites and hormones in fetal plasma, amino acid concentrations in the fetal liver and longissimus dorsi muscle, and expression of mRNAs in the somatotropic axis. Maternal and fetal bodyweight and the weight of most fetal organs expressed as a percentage of bodyweight increased in response to ARG and NCG compared with values for fetuses from RG ewes. Fetal plasma concentrations of insulin, insulin-like growth factor 1, total amino acids, lactate, thyroxine, and the thyroxine/tri-iodothyronine ratio were lower in fetuses from RG ewes compared with the other treatment groups, but concentrations of growth hormone, non-esterified fatty acids, and total cholesterol were greater in fetuses from RG ewes. Maternal RP-Arg or NCG supplementation increased concentrations of amino acids in fetal tissues and expression of mRNAs for somatotropic axis proteins in fetuses from RG ewes. These findings suggest that maternal RP-Arg and NCG supplementation of underfed ewes decreases fetal IUGR by improving metabolic homeostasis of fetal endocrinology, increasing the availability of amino acids in the fetal liver and longissimus dorsi muscle and affecting the expression of somatotropic axis genes.

N-carbamylglutamate and L-arginine improved maternal and placental development in underfed ewes.

The objectives of this study were to determine how dietary supplementation of N-carbamylglutamate (NCG) and rumen-protected L-arginine (RP-Arg) in nutrient-restricted pregnant Hu sheep would affect (1) maternal endocrine status; (2) maternal, fetal, and placental antioxidation capability; and (3) placental development. From day 35 to day 110 of gestation, 32 Hu ewes carrying twin fetuses were allocated randomly into four groups: 100% of NRC-recommended nutrient requirements, 50% of NRC recommendations, 50% of NRC recommendations supplemented with 20g/day RP-Arg, and 50% of NRC recommendations supplemented with 5g/day NCG product. The results showed that in maternal and fetal plasma and placentomes, the activities of total antioxidant capacity and superoxide dismutase were increased (P<0.05); however, the activity of glutathione peroxidase and the concentration of maleic dialdehyde were decreased (P<0.05) in both NCG- and RP-Arg-treated underfed ewes. The mRNA expression of vascular endothelial growth factor and Fms-like tyrosine kinase 1 was increased (P<0.05) in 50% NRC ewes than in 100% NRC ewes, and had no effect (P>0.05) in both NCG- and RP-Arg-treated underfed ewes. A supplement of RP-Arg and NCG reduced (P<0.05) the concentrations of progesterone, cortisol, and estradiol-17ß; had no effect on T4/T3; and improved (P<0.05) the concentrations of leptin, insulin-like growth factor 1, tri-iodothyronine (T3), and thyroxine (T4) in serum from underfed ewes. These results indicate that dietary supplementation of NCG and RP-Arg in underfed ewes could influence maternal endocrine status, improve the maternal-fetal-placental antioxidation capability, and promote fetal and placental development during early-to-late gestation.

(1)H-Nuclear Magnetic Resonance-Based Plasma Metabolic Profiling of Dairy Cows with Fatty Liver.

Fatty liver is a common metabolic disorder of dairy cows during the transition period. Historically, the diagnosis of fatty liver has involved liver biopsy, biochemical or histological examination of liver specimens, and ultrasonographic imaging of the liver. However, more convenient and noninvasive methods would be beneficial for the diagnosis of fatty liver in dairy cows. The plasma metabolic profiles of dairy cows with fatty liver and normal (control) cows were investigated to identify new biomarkers using (1)H nuclear magnetic resonance. Compared with the control group, the primary differences in the fatty liver group included increases in ß-hydroxybutyric acid, acetone, glycine, valine, trimethylamine-N-oxide, citrulline, and isobutyrate, and decreases in alanine, asparagine, glucose, γ-aminobutyric acid glycerol, and creatinine. This analysis revealed a global profile of endogenous metabolites, which may present potential biomarkers for the diagnosis of fatty liver in dairy cows.

Dietary Supplementation with Rumen-Protected Arginine or N-Carbamylglutamate Enhances Fetal Liver Development in Nutrient-Restricted Pregnant Hu Ewes.

This study was conducted in nutrient-restricted pregnant Hu ewes to determine whether rumen-protected arginine (RP-Arg) or N-carbamylglutamate (NCG) supplementation affects fetal liver growth and development. From 35 d to 110 d of gestation, 32 Hu ewes were randomly divided into four groups: a control group (100% of the National Research Council (NRC) requirements), a nutrient-restricted group (50% of the NRC requirements), and two treatment groups (ARG and NCG, 50% of the NRC requirements, supplemented with 20 g/day RP-Arg or 5 g/day NCG, respectively). Fetal body weights, fetal liver growth performance, the capability of antioxidation, and the expression of the mRNA and proteins of apoptosis-related genes in the fetal liver were determined and analyzed at 110 d of gestation. The dry matter, water, fat, protein, and ash components of the fetal livers in the RG group were found to be lower than in the CG group, and these components were significantly higher in the NCG group than in the RG group (p < 0.05). A decrease in DNA, RNA, and protein concentrations and contents, as well as in protein/DNA ratios, was observed in the RG group in comparison to the CG group (p < 0.05). Compared with the RG group, the NCG group had higher concentrations of DNA, RNA, and protein, as well as higher protein/DNA ratios (p < 0.05). The RG group had lower concentrations of cholinesterase, nitric oxide, nitric oxide synthase, superoxide dismutase, alanine aminotransferase, and total protein than the CG group (p < 0.05). The RG group had higher levels of glutathione peroxidase, maleic dialdehyde, and aspartate aminotransferase than the CG group (p < 0.05). In the RG group, the mRNA and protein expression of p53 and Bax was significantly increased (p < 0.05) compared with the CG group, and the gene expression of FasL and Bcl-2, the ratio of Bcl-2 to Bax, and the protein expression of Bcl-2 in the RG group were lower (p < 0.05) than in the CG group. It appears that RP-Arg and NCG supplementation during pregnancy could influence fetal liver growth and development. A nutrition-based therapeutic intervention to alleviate reduced fetal growth can be developed based on this study, which has demonstrated that maternal undernutrition during pregnancy induces the maldevelopment of the fetal liver.

Characterization and Comparative Analysis of Whole-Transcriptome Sequencing in High- and Low-Fecundity Chongming White Goat Ovaries during the Estrus Phase.

Reproductive performance is one of the most important economic traits in the goat industry. Increasing the number of goats is an effective measure to improve production efficiency and reduce production costs. Ovaries are important reproductive organs in female mammals that directly affect the estrous cycle and reproductive abilities. Understanding the complex transcription network of non-coding RNAs (lncRNAs, circRNAs, and miRNAs) and messenger RNA (mRNA) could lead to significant insights into the ovarian regulation of the reproductive processes of animals. However, the whole-transcriptome analysis of the non-coding RNAs and mRNA of the ovaries in Chongming white goats between high-fecundity (HP) and low-fecundity (LP) groups is limited. In this study, a whole-transcriptome sequencing approach was used to identify lncRNA, circRNA, miRNA, and mRNA expression in the ovaries of Chongming white goats during the estrus phase using RNA-Seq technology. More than 20,000 messenger RNAs (mRNAs), 10,000 long non-coding RNAs (lncRNAs), 3500 circular RNAs (circRNAs), and 1000 micro RNAs (miRNAs) were identified. A total of 1024 differential transcripts (724 mRNAs, 112 lncRNAs, 178 circRNAs, and 10 miRNAs) existing between the HP and the LP groups were revealed through a bioinformatics analysis. They were enriched in the prolactin signaling pathway, the Jak-STAT signaling pathway, and the GnRH signaling pathway, as well as various metabolic pathways. Differentially expressed mRNAs (such as LYPD6, VEGFA, NOS3, TNXB, and EPHA2) and miRNAs (such as miR-10a-5p) play key roles in the regulation of goat ovaries during the estrus phase. The enrichment of pathways related to reproduction, such as the Hippo, Hedgehog, PI3K-AKT, and MAPK signaling pathways, suggests that they might be involved in the prolificacy of goat ovaries. Overall, we identified several gene modules associated with goat fecundity and provided a basis for a molecular mechanism in the ovaries of Chongming white goats.

Characterization of Meat Metabolites and Lipids in Shanghai Local Pig Breeds Revealed by LC-MS-Based Method.

The meat of local livestock breeds often has unique qualities and flavors. In this study, three Shanghai native pig breeds (MSZ, SWT, and SHB) exhibited better meat quality traits than globalized commercial pig breeds (DLY). Subsequently, metabolomic and lipidomic differences in the longissimus dorsi (L) and gluteus (T) muscles of the Shanghai native pig breeds and DLY pig breed were compared using liquid chromatography-mass spectrometry (LC-MS). The results demonstrated that the metabolites mainly consisted of (28.16%) lipids and lipid-like molecules, and (25.87%) organic acids and their derivatives were the two most dominant groups. Hundreds of differential expression metabolites were identified in every compared group, respectively. One-way ANOVA was applied to test the significance between multiple groups. Among the 20 most abundant differential metabolites, L-carnitine was significantly different in the muscles of the four pig breeds (p-value = 7.322 × 10-11). It was significantly higher in the L and T muscles of the two indigenous black pig breeds (MSZ and SWT) than in the DLY pigs (p-value < 0.001). Similarly, lipidomic analysis revealed the PA (18:0/18:2) was significantly more abundant in the muscle of these two black breeds than that in the DLY breed (p-value < 0.001). These specific metabolites and lipids might influence the meat quality and taste properties and lead to customer preferences. Therefore, this study provided insights into the characterization of meat metabolites and lipids in Shanghai native pig breeds.

Detection and Analysis of Antidiarrheal Genes and Immune Factors in Various Shanghai Pig Breeds.

The aim of this study was to identify effective genetic markers for the Antigen Processing Associated Transporter 1 (TAP1), α (1,2) Fucosyltransferase 1 (FUT1), Natural Resistance Associated Macrophage Protein 1 (NRAMP1), Mucin 4 (MUC4) and Mucin 13 (MUC13) diarrhea-resistance genes in the local pig breeds, namely Shanghai white pigs, Fengjing pigs, Shawutou pigs, Meishan pigs and Pudong white pigs, to provide a reference for the characterization of local pig breed resources in Shanghai. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLR) and sequence sequencing were applied to analyze the polymorphisms of the above genes and to explore the effects on the immunity of Shanghai local pig breeds in conjunction with some immunity factors. The results showed that both TAP1 and MUC4 genes had antidiarrheal genotype GG in the five pig breeds, AG and GG genotypes of the FUT1 gene were detected in Pudong white pigs, AA antidiarrheal genes of the NRAMP1 gene were detected in Meishan pigs, the AB type of the NRAMP1 gene was detected in Pudong white pigs, and antidiarrheal genotype GG of the MUC13 gene was only detected in Shanghai white pigs. The MUC13 antidiarrhea genotype GG was only detected in Shanghai white pigs. The TAP1 gene was moderately polymorphic in Shanghai white pigs, Fengjing pigs, Shawutou pigs, Meishan pigs and Pudong white pigs, among which TAP1 in Shanghai white pigs and Shawutou pigs did not satisfy the Hardy-Weinberg equilibrium. The FUT1 gene of Pudong white pigs was in a state of low polymorphism. NRAMP1 of Meishan pigs and Pudong white pigs was in a state of moderate polymorphism, which did not satisfy the Hardy-Weinberg equilibrium. The MUC4 genes of Shanghai white pigs and Pudong white pigs were in a state of low polymorphism, and the MUC4 genes of Fengjing pigs and Shawutou pigs were in a state of moderate polymorphism, and the MUC4 genes of Fengjing pigs and Pudong white pigs did not satisfy the Hardy-Weinberg equilibrium. The MUC13 gene of Shanghai white pigs and Pudong white pigs was in a state of moderate polymorphism. Meishan pigs had higher levels of IL-2, IL-10, IgG and TNF-α, and Pudong white pigs had higher levels of IL-12 than the other pigs. The level of interleukin 12 (IL-12) was significantly higher in the AA genotype of the MUC13 gene of Shanghai white pigs than in the AG genotype. The indicator of tumor necrosis factor alpha (TNF-α) in the AA genotype of the TAP1 gene of Fengjing pigs was significantly higher than that of the GG and AG genotypes. The indicator of IL-12 in the AG genotype of the Shawutou pig TAP1 gene was significantly higher than that of the GG genotype. The level of TNF-α in the AA genotype of the NRAMP1 gene of Meishan pigs was markedly higher than that of the AB genotype. The IL-2 level of the AG type of the FUT1 gene was obviously higher than that of the GG type of Pudong white pigs, the IL-2 level of the AA type of the MUC4 gene was dramatically higher than that of the AG type, and the IgG level of the GG type of the MUC13 gene was apparently higher than that of the AG type. The results of this study are of great significance in guiding the antidiarrhea breeding and molecular selection of Shanghai white pigs, Fengjing pigs, Shawutou pigs, Meishan pigs and Pudong white pigs and laying the foundation for future antidiarrhea breeding of various local pig breeds in Shanghai.

Evaluation of Dry Ice for Short-Term Storage and Transportation of Frozen Boar Semen.

To address the safety problems posed by the transportation of boar semen using LN, this study was conducted on the short-term storage of frozen boar semen in dry ice (-79 °C). Boar semen frozen in LN was transferred to dry ice, kept for 1 day, 3 days, 5 days, 7 days, or 8 days, and then moved back to LN. The quality of frozen semen stored in LN or dry ice was determined to evaluate the feasibility of short-distance transportation with dry ice. The results showed that 60 °C for 8 s was the best condition for thawing frozen semen stored in dry ice. No significant differences in spermatozoa motility, plasma membrane integrity, or acrosome integrity were observed in semen after short-term storage in dry ice compared to LN (p > 0.05). There were no significant changes in antioxidant properties between storage groups either (p > 0.05). In conclusion, dry ice could be used as a cold source for the short-term transportation of frozen boar semen for at least 7 days, without affecting sperm motility, morphological integrity, or antioxidant indices.

Plasma metabolomic profiling of dairy cows affected with ketosis using gas chromatography/mass spectrometry.

BACKGROUND: Ketosis is an important problem for dairy cows` production performance. However, it is still little known about plasma metabolomics details of dairy ketosis. RESULTS: A gas chromatography/mass spectrometry (GC/MS) technique was used to investigate plasma metabolic differences in cows that had clinical ketosis (CK, n=22), subclinical ketosis (SK, n=32), or were clinically normal controls (NC, n=22). The endogenous plasma metabolome was measured by chemical derivatization followed by GC/MS, which led to the detection of 267 variables. A two-sample t-test of 30, 32, and 13 metabolites showed statistically significant differences between SK and NC, CK and NC, and CK and SK, respectively. Orthogonal signal correction-partial least-square discriminant analysis (OPLS-DA) revealed that the metabolic patterns of both CK and SK were mostly similar, with the exception of a few differences. The development of CK and SK involved disturbances in many metabolic pathways, mainly including fatty acid metabolism, amino acid metabolism, glycolysis, gluconeogenesis, and the pentose phosphate pathway. A diagnostic model arbitrary two groups was constructed using OPLS-DA and receiver-operator characteristic curves (ROC). Multivariate statistical diagnostics yielded the 19 potential biomarkers for SK and NC, 31 for CK and NC, and 8 for CK and SK with area under the curve (AUC) values. Our results showed the potential biomarkers from CK, SK, and NC, including carbohydrates, fatty acids, amino acids, even sitosterol and vitamin E isomers, etc. 2-piperidinecarboxylic acid and cis-9-hexadecenoic acid were closely associated with metabolic perturbations in ketosis as Glc, BHBA and NEFA for dealing with metabolic disturbances of ketosis in clinical practice. However, further research is needed to explain changes of 2,3,4-trihydroxybutyric acid, 3,4-dihydroxybutyric acid, α-aminobutyric acid, methylmalonic acid, sitosterol and α-tocopherol in CK and SK, and to reveal differences between CK and SK. CONCLUSION: Our study shows that some new biomarkers of ketosis from plasma may find new metabolic changes to have clinically new utility and significance in diagnosis, prognosis, and prevention of ketosis in the future.

Multiple independent loci at chromosome 15q25.1 affect smoking quantity: a meta-analysis and comparison with lung cancer and COPD.

Recently, genetic association findings for nicotine dependence, smoking behavior, and smoking-related diseases converged to implicate the chromosome 15q25.1 region, which includes the CHRNA5-CHRNA3-CHRNB4 cholinergic nicotinic receptor subunit genes. In particular, association with the nonsynonymous CHRNA5 SNP rs16969968 and correlates has been replicated in several independent studies. Extensive genotyping of this region has suggested additional statistically distinct signals for nicotine dependence, tagged by rs578776 and rs588765. One goal of the Consortium for the Genetic Analysis of Smoking Phenotypes (CGASP) is to elucidate the associations among these markers and dichotomous smoking quantity (heavy versus light smoking), lung cancer, and chronic obstructive pulmonary disease (COPD). We performed a meta-analysis across 34 datasets of European-ancestry subjects, including 38,617 smokers who were assessed for cigarettes-per-day, 7,700 lung cancer cases and 5,914 lung-cancer-free controls (all smokers), and 2,614 COPD cases and 3,568 COPD-free controls (all smokers). We demonstrate statistically independent associations of rs16969968 and rs588765 with smoking (mutually adjusted p-values<10(-35) and <10(-8) respectively). Because the risk alleles at these loci are negatively correlated, their association with smoking is stronger in the joint model than when each SNP is analyzed alone. Rs578776 also demonstrates association with smoking after adjustment for rs16969968 (p<10(-6)). In models adjusting for cigarettes-per-day, we confirm the association between rs16969968 and lung cancer (p<10(-20)) and observe a nominally significant association with COPD (p = 0.01); the other loci are not significantly associated with either lung cancer or COPD after adjusting for rs16969968. This study provides strong evidence that multiple statistically distinct loci in this region affect smoking behavior. This study is also the first report of association between rs588765 (and correlates) and smoking that achieves genome-wide significance; these SNPs have previously been associated with mRNA levels of CHRNA5 in brain and lung tissue.

Genetics of vegetarianism: A genome-wide association study.

A substantial body of evidence points to the heritability of dietary preferences. While vegetarianism has been practiced for millennia in various societies, its practitioners remain a small minority of people worldwide, and the role of genetics in choosing a vegetarian diet is not well understood. Dietary choices involve an interplay between the physiologic effects of dietary items, their metabolism, and taste perception, all of which are strongly influenced by genetics. In this study, we used a genome-wide association study (GWAS) to identify loci associated with strict vegetarianism in UK Biobank participants. Comparing 5,324 strict vegetarians to 329,455 controls, we identified one SNP on chromosome 18 that is associated with vegetarianism at the genome-wide significant level (rs72884519, ß = -0.11, P = 4.997 x 10-8), and an additional 201 suggestively significant variants. Four genes are associated with rs72884519: TMEM241, RIOK3, NPC1, and RMC1. Using the Functional Mapping and Annotation (FUMA) platform and the Multi-marker Analysis of GenoMic Annotation (MAGMA) tool, we identified 34 genes with a possible role in vegetarianism, 3 of which are GWAS-significant based on gene-level analysis: RIOK3, RMC1, and NPC1. Several of the genes associated with vegetarianism, including TMEM241, NPC1, and RMC1, have important functions in lipid metabolism and brain function, raising the possibility that differences in lipid metabolism and their effects on the brain may underlie the ability to subsist on a vegetarian diet. These results support a role for genetics in choosing a vegetarian diet and open the door to future studies aimed at further elucidating the physiologic pathways involved in vegetarianism.

Genomic Dissection through Whole-Genome Resequencing of Five Local Pig Breeds from Shanghai, China.

China has rich genetic resources of local pig breeds. In this study, whole-genome resequencing was performed on five Shanghai local pig breeds, aiming to analyze their population genetic structure and unique genomic characteristics. Tens of millions of single nucleotide variants were obtained through the resequencing of a total of 150 individual pigs from five local pig breeds (Meishan, Fengjing, Shawutou, Pudong White, and Shanghai White) after mapping them with the pig reference genome of Sus scrofa 11.1. The results of admixture structure analysis also clearly demonstrated the genetic differences between the Shanghai local pig breeds and the three commercial pig breeds (Duroc, Landrace, and Yorkshire). The genetic infiltration of Landrace and Yorkshire pig breeds in the SHW breed was detected, which is consistent with the early history of crossbreeding in this breed. Selective sweep analysis between four indigenous Shanghai pig breed populations and three commercial pig breed populations identified 270 and 224 genes with selective signatures in the commercial and indigenous Shanghai pig populations, respectively. Six genes (TGS1, PLAG1, CHCHD7, LCORL, TMEM68, and TMEM8B) were found to be associated with animal growth in the commercial pig population through gene enrichment and protein-protein interaction analysis. In contrast, the MSRB3 gene in the indigenous Shanghai pig population was significantly under selection, which correlated with the long pendulous ear phenotype of the indigenous Shanghai pig population. In conclusion, this study is the first genomic profiling of five representative local pig breeds in Shanghai, which provides molecular genetic data and foundations for better conservation and utilization of local pig breed resources in Shanghai, China.

Effect of Poria cocos Mushroom Polysaccharides (PCPs) on the Quality and DNA Methylation of Cryopreserved Shanghai White Pig Spermatozoa.

In this study, we explore the effects of Poria cocos mushroom polysaccharides (PCPs) on the quality and DNA methylation of the cryopreserved spermatozoa of Shanghai white pigs. A total of 24 ejaculates (three ejaculate samples per boar) from eight Shanghai white pigs were manually collected. The pooled semen was diluted with a based extender supplemented with different concentrations of PCPs (0, 300, 600, 900, 1200, and 1500 µg/mL). Once thawed, the quality of the spermatozoa and their antioxidant function were assessed. In the meantime, the effect of spermatozoa DNA methylation was also analyzed. The results show that compared with the control group, 600 µg/mL of PCPs significantly improves the spermatozoa viability (p < 0.05). The motility and plasma membrane integrity of the frozen-thawed spermatozoa are significantly higher after treatment with 600, 900, and 1200 µg/mL of PCPs compared with the control group (p < 0.05). In comparison with the control group, the percentages of acrosome integrity and mitochondrial activity are significantly enhanced after the application of 600 and 900 µg/mL PCPs (p < 0.05). The reactive oxygen species (ROS), the malondialdehyde (MDA) levels, and the glutathione peroxidase (GSH-Px) activity, in comparison with the control group, are significantly decreased in all groups with PCPs (all p < 0.05). The enzymatic activity of superoxide dismutase (SOD) in spermatozoa is significantly higher in the treatment with 600 µg/mL of PCPs than in the other groups (p < 0.05). As compared with the control group, a significant increase in the catalase (CAT) level is found in the groups with PCPs at 300, 600, 900, and 1200 µg/mL (all p < 0.05). In comparison with the control group, the 5-methylcytosine (5-mC) levels are significantly decreased in all groups with PCPs (all p < 0.05). As a result of these findings, a certain amount of PCPs (600-900 µg/mL) added to the cryodiluent can significantly improve the quality of Shanghai white pig spermatozoa and can also reduce the methylation of spermatozoa DNA caused by cryopreservation. This treatment strategy may establish a foundation for the cryopreservation of semen from pigs.

Comparative Cytotoxic Effects and Possible Mechanisms of Deoxynivalenol, Zearalenone and T-2 Toxin Exposure to Porcine Leydig Cells In Vitro.

Mycotoxins such as zearalenone (ZEN), deoxynivalenol (DON) and T-2 toxin (T-2) are the most poisonous biological toxins in food pollution. Mycotoxin contaminations are a global health issue. The aim of the current study was to use porcine Leydig cells as a model to explore the toxic effects and underlying mechanisms of ZEN, DON and T-2. The 50% inhibitory concentration (IC50) of ZEN was 49.71 µM, and the IC50 values of DON and T-2 were 2.49 µM and 97.18 nM, respectively. Based on the values of IC50, ZEN, DON and T-2 exposure resulted in increased cell apoptosis, as well as disrupted mitochondria membrane potential and cell cycle distribution. The results also showed that ZEN and DON significantly reduced testosterone and progesterone secretion in Leydig cells, but T-2 only reduced testosterone secretion. Furthermore, the expression of steroidogenic acute regulatory (StAR) protein and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) were significantly decreased by ZEN, DON and T-2; whereas the protein expression of cholesterol side-chain cleavage enzyme (CYP11A1) was only significantly decreased by ZEN. Altogether, these data suggest that the ZEN, DON and T-2 toxins resulted in reproductive toxicity involving the inhibition of steroidogenesis and cell proliferation, which contributes to the cellular apoptosis induced by mitochondrial injury in porcine Leydig cells.

Screening Discriminating SNPs for Chinese Indigenous Pig Breeds Identification Using a Random Forests Algorithm.

Chinese indigenous pig breeds have unique genetic characteristics and a rich diversity; however, effective breed identification methods have not yet been well established. In this study, a genotype file of 62,822 single-nucleotide polymorphisms (SNPs), which were obtained from 1059 individuals of 18 Chinese indigenous pig breeds and 5 cosmopolitan breeds, were used to screen the discriminating SNPs for pig breed identification. After linkage disequilibrium (LD) pruning filtering, this study excluded 396 SNPs on non-constant chromosomes and retained 20.92~-27.84% of SNPs for each of the 18 autosomes, leaving a total of 14,823 SNPs. The principal component analysis (PCA) showed the largest differences between cosmopolitan and Chinese pig breeds (PC1 = 10.452%), while relatively small differences were found among the 18 indigenous pig breeds from the Yangtze River Delta region of China. Next, a random forest (RF) algorithm was used to filter these SNPs and obtain the optimal number of decision trees (ntree = 1000) using corresponding out-of-bag (OOB) error rates. By comparing two different SNP ranking methods in the RF analysis, the mean decreasing accuracy (MDA) and mean decreasing Gini index (MDG), the effects of panels with different numbers of SNPs on the assignment accuracy, and the statistics of SNP distribution on each chromosome in the panels, a panel of 1000 of the most breed-discriminative tagged SNPs were finally selected based on the MDA screening method. A high accuracy (>99.3%) was obtained by the breed prediction of 318 samples in the RF test set; thus, a machine learning classification method was established for the multi-breed identification of Chinese indigenous pigs based on a low-density panel of SNPs.