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.

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.

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.

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.

Does Antioxidant Mitoquinone (MitoQ) Ameliorate Oxidative Stress in Frozen-Thawed Rooster Sperm?

In this study, we aimed to determine the benefit of mitoquinone (MitoQ) in rooster semen extenders on sperm quality, motility parameters, antioxidant capacities, and apoptotic changes in post-thawed rooster semen. A total of 85 ejaculates from 18 roosters were collected and then divided into five equal aliquots and cryopreserved in extenders with 1.0% soy lecithin nanoparticles that contained various concentrations of MitoQ (0 nM (M0), 50 nM (M50), 100 nM (M100), 150 nM (M150), and 200 nM (M200)). By using a computer-assisted semen analyzer, sperm motility parameters were assessed after freeze thawing. The M150 group had significantly higher percentages of total motility, progressive motility, viability, acrosome membrane integrity, and mitochondrial activity than the other groups (p < 0.05). Compared to other groups, M100 and M150 groups produced a higher percentage of plasma membrane integrity and ATP contents (p < 0.05). Additionally, the lowest levels of ROS and MDA in spermatozoa were observed in M150 group (p < 0.05), whereas the highest levels of ROS and MDA were observed in sperm in the controls or the M200 group (p < 0.05). Significantly higher values of SOD, GPx, and Cas-3 were found in the M150 group compared to other groups (p < 0.05). Overall, these results demonstrate that MitoQ at 150 nM not only ameliorates post-thawed sperm quality and motility parameters by restoring ATP levels and preventing membrane damage, but also improves redox balance and antiapoptotic activities.

Resveratrol Protects against Zearalenone-Induced Mitochondrial Defects during Porcine Oocyte Maturation via PINK1/Parkin-Mediated Mitophagy.

Mitochondria hold redox homeostasis and energy metabolism as a crucial factor during oocyte maturation, while the exposure of estrogenic mycotoxin zearalenone causes developmental incapacity in porcine oocyte. This study aimed to reveal a potential resistance of phytoalexin resveratrol against zearalenone during porcine oocyte maturation and whether its mechanism was related with PTEN-induced kinase 1 (PINK1)/Parkin-mediated mitophagy. Porcine oocytes were exposed to 20 µM zearalenone with or without 2 µM resveratrol during in vitro maturation. As for the results, zearalenone impaired ultrastructure of mitochondria, causing mitochondrial depolarization, oxidative stress, apoptosis and embryonic developmental incapacity, in which mitophagy was induced in response to mitochondrial dysfunction. Phytoalexin resveratrol enhanced mitophagy through PINK1/Parkin in zearalenone-exposed oocytes, manifesting as enhanced mitophagy flux, upregulated PINK1, Parkin, microtubule-associated protein light-chain 3 beta-II (LC3B-II) and downregulated substrates mitofusin 2 (MFN2), voltage-dependent anion channels 1 (VDAC1) and p62 expressions. Resveratrol redressed zearalenone-induced mitochondrial depolarization, oxidative stress and apoptosis, and accelerated mitochondrial DNA copy during maturation, which improved embryonic development. This study offered an antitoxin solution during porcine oocyte maturation and revealed the involvement of PINK1/Parkin-mediated mitophagy, in which resveratrol mitigated zearalenone-induced embryonic developmental incapacity.

Sema6D Regulates Zebrafish Vascular Patterning and Motor Neuronal Axon Growth in Spinal Cord.

Vessels and nerves are closely associated in anatomy as well as functions. Accumulating evidences have demonstrated that axon-guiding signals may affect endothelial cells migration and path finding, which is crucial for the patterning of both the complex vascular network and neural system. However, studies regarding the functional overlap between vascular and neuronal orchestrating are still incomplete. Semaphorin6D (Sema6D) belongs to the Semaphorin family and has been identified as an important regulating factor in diverse biological processes. Its roles in vascular development are still unclear. Here, we confirmed that sema6D is enriched in neural system and blood vessels of zebrafish embryos by in situ hybridization. Then, the deficiency of sema6D caused by specific antisense morpholino-oligonucleotides (MO) led to dramatic path finding defects in both intersegmental vessels (ISVs) and primary motor neurons (PMNs) of spinal cord in zebrafish embryos. Furthermore, these defective phenotypes were confirmed in F0 generation of sema6D knockouts and rescue experiments by overexpression of sema6D mRNA in sema6D morphants. These data collectively indicate that sema6D regulates zebrafish vascular patterning and motor neuronal axon growth in the spinal cord, which might be of great therapeutical use to regulate vessel and nerve guidance in the relevant diseases that affect both systems.

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.

Involvement of PINK1/Parkin-mediated mitophagy in mitochondrial functional disruption under oxidative stress in vitrified porcine oocytes.

Vitrification is an effective technique for fertility preservation, but is known to lead to mitochondrial dysfunction in porcine oocytes. Mitophagy is induced to rebalance mitochondrial function, a process in which reactive oxygen species (ROS) plays a role. In this study, vitrified-warmed porcine oocytes were incubated for 4 h with the oxidant AAPH or antioxidant α-tocopherol to alter ROS levels. A series of tests suggested that vitrification damaged mitochondrial structure and caused dysfunction, including blurred mitochondrial cristae, decreased mitochondrial membrane potential, decreased mtDNA copy number and increased ROS generation. This dysfunction resulted in mitophagy and the loss of embryonic developmental potential. Incubation with AAPH or α-tocopherol altered mitochondrial function and mitophagy flux status in vitrified oocytes. The PINK1/Parkin pathway was involved in oxidative stress regulation in vitrified oocytes. Under AAPH-induced oxidative stress, increased fluorescence intensity of Parkin, increased expression of PINK1, Parkin, and LC3B-II, and decreased expression of MFN2 and p62 were observed, whereas the opposite effects were induced under α-tocopherol treatment. The inhibition of ROS by α-tocopherol benefitted mitochondrial homeostasis and alleviated PINK1/Parkin-mediated mitophagy, resulting in the recovery of embryonic developmental potential in vitrified porcine oocytes. Therefore, this study provides a new mechanism for the application of antioxidants to aid the cryopreservation of porcine oocytes.

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.