Bama Minipigs Research Articles

Nanoemulsion-based transdermal delivery of third-generation steroidal and non-steroidal aromatase inhibitors in preclinical models.

Aromatase inhibitors are effective in treating hormone receptor-positive breast cancer, particularly in postmenopausal women. However, the challenges of inconsistent dissolution, variable absorption and side effects with oral administration persist. To address these issues, transdermal delivery has emerged as a viable alternative. In our study, we have developed nanoemulsion-based transdermal creams containing third-generation aromatase inhibitors Exemestane (EXE) or Letrozole (LE) and evaluated their toxicity, anti-tumour effects and androgenic potency using preclinical models including Bama minipigs, DMBA-induced breast cancer rats and orchidectomized male rats. The results of our study are significant, suggesting that both creams effectively penetrated the skin, demonstrating an impressive anti-breast cancer effect. Importantly, EXE cream had no organ toxicity at the tested dose, providing a reassuring safety profile for its use. In contrast, LE cream displayed reversible toxicity from drug molecule itself in animals at the given dose, dissipating after 3 weeks of withdrawal and recovery. This study establishes a solid foundation for the safe clinical use of third-generation aromatase inhibitors. It highlights transdermal creams as a promising drug delivery carrier for administering them.

Characterization of three African swine fever viruses from different clinical settings revealed a potential attenuation mechanism

African swine fever (ASF) is an acute and fatal hemorrhagic disease in domestic pigs and wild boars caused by African swine fever virus (ASFV) that currently threatens the pig industry worldwide. Since the 2018 ASF outbreak in China, ASFV has evolved and caused diverse clinical manifestations, such as chronic and asymptomatic infections. Therefore, it is important to understand the molecular mechanisms underlying ASFV attenuation in the field. Here, we isolated three ASFVs from one diseased and two asymptomatic pigs by using primary porcine alveolar macrophages (PAMs) from both domestic pigs and Bama minipigs. The three ASFVs exhibited similar phenotypes in cell culture, including cytopathic effects (CPEs), hemadsorptions (HADs), viral protein expressions and growth curves. Genome sequencing revealed that all three ASFVs were genotype II strains. Genomic comparisons suggested that the disruption of the viral genes MGF360 and MGF110, rather than EP402R and EP153R, is likely involved in the potential attenuation of ASFV via the upregulation of innate immune responses.

Expression mechanisms of mir-486-5p and its host gene sANK1 in porcine muscle.

MiR-486-5p has been identified as a crucial regulator of the PI3K/AKT signalling pathway, which plays a significant role in skeletal muscle development. Its host gene, sANK1, is also essential for skeletal muscle development. However, the understanding of porcine miR-486-5p and sANK1 has been limited. In this study, PCR analyses revealed a positive correlation between the expression of miR-486-5p and sANK1 in the longissimus dorsi muscle of the Bama mini-pig and Landrace-pig, as well as during myoblast differentiation. Furthermore, the expression of miR-486-5p/sANK1 was higher in the Bama mini-pig compared to the Landrace-pig. There was a total of 18 single nucleotide polymorphisms (SNP) present in the sANK1 promoter region. Among these SNPs, 14 of them resulted in alterations in transcription factor binding sites (TFBs). Additionally, the promoter fluorescence assay demonstrated that the activity of the sANK1 promoter derived from the Bama mini-pig was significantly higher compared to Landrace-pig. It is worth noting that ten regulatory SNPs have the potential to influence the activity of the sANK1 promoter. A nuclear mutation A-G located at position - 401 (relative to the transcription start site) in the Bama mini-pig was identified, which creates a putative TFB motif for MyoD. The findings presented in this study offer fundamental molecular knowledge and expression patterns of miR-486-5p/sANK1, which can be valuable for gaining a deeper understanding of the gene's involvement in porcine skeletal muscle development, and meat quality.

Biological performance of a bioabsorbable magnesium-magnesium phosphate cement interbody fusion cage in a porcine lumbar interbody fusion model: a feasibility study.

The aim of the study was to evaluate the feasibility of a bioabsorbable cage consisting of magnesium and magnesium phosphate cement (MPC) in a porcine lumbar interbody fusion model. Twelve male Ba-Ma mini pigs underwent lumbar discectomy and fusion with an Mg-MPC cage or a PEEK cage at the L3/L4 and L4/L5 level. Computed tomography (CT) scans were made to evaluate the distractive property by comparing average disc space height (DSH) before and at 6, 12, and 24 weeks after the operation. After the lumbar spines were harvested at 6 or 24 weeks after the operation, micro-CT examination was conducted to analyze the fusion rate, and stiffness of motion segments was investigated through mechanical tests. A histological study was performed to evaluate the tissue type, inflammation, and osteolysis in the intervertebral space. CT scans showed no significant difference between the two groups in average DSH at each time point. Micro-CT scans revealed an equal fusion rate in both groups (0% at 6 weeks, 83.3% at 24 weeks). Both groups showed time-dependent increases in stability, the Mg-MPC cages achieved an inferior stiffness at 6 weeks and a comparable stiffness at 24 weeks. Histologic evaluation showed the presence of newly formed bone in both groups. However, empty spaces were observed at the interface or around the Mg-MPC cages. Compared with the PEEK cages, the Mg-MPC cages achieved comparable distraction, fusion rate, and spinal stability at 24 weeks after the operation. However, due to inferior stiffness at the early stage and fast degradation, further modification of material composition and design are necessary.

Study on the application of self-fixing and self-detachable drainage stent in pancreaticojejunostomy

Objectives: To evaluate the efficacy and safety of the self-fixing and self-detachable drainage stent in pancreaticojejunostomy and to provide supportive data for the follow clinical trials. Methods: This is an experimental research in animals which completed from February 2022 to September 2022. A self-fixing and self-detachable pancreaticojejunostomy drainage stent was designed for Hong's pancreaticojejunostomy technique based on the theory of "fistula healing" in pancreaticojejunostomy. Ten biocompatibility tests were completed in vitro before this study. Twenty-five Bama minipigs were selected and double-ligated in the neck of the pancreas to dilate the distal main pancreatic duct. Twenty-three of them were successfully modelled and divided into three groups by a stratified random method: pancreaticojejunostomy drainage stent group (referred to as stent group) with 11 pigs, pancreatic duct to jejunal mucosa anastomosis group (referred to as manual suture group) with 8 pigs, sham operation group with 4 pigs. The anastomic time,amylase content in postoperative abdominal drainage fluid and the tolerable pressure value of pancreaticojejunostomy were compared between the stent group and the manual suture group. An abdominal X-ray fluoroscopy examination was adopted to detect the detach time of the stent. A postoperative pathological examination was performed to verify the healing time,the type of treatment and the stricture rate of pancreaticojejunostomy. Quantitative data was analyzed by independent sample t-test. The classified data were analyzed by Fisher's exact test. Results: There were no significant differences in the diameter of the pancreatic duct and pancreatic texture,the time of pancreaticojejunostomy,the amylase content in postoperative peritoneal drainage fluid,and the tolerable pressure value of the pancreaticojejunostomy between the stent group and the manual suture group(all P>0.05). Abdominal X-ray fluoroscopy showed that the stents gradually detached and were removed from the body 21 days after operation,and all stents were detached in the follow 3 months after operation. Pancreaticojejunostomy healed 7 days after operation based on fistula formation in the stent group,and 14 days in the manual suture group. The incidence of anastomotic stricture within 35 days after operation was 2/8 in the stent group and 6/8 in the manual suture group (Fisher's exact test: P=0.132). Conclusion: The stent method is safer and simpler than the manual suture method in pancreaticojejunostomy of Bama minipigs, with shorter anastomotic healing time and lower stricture rate.

Injectable chitosan-polyvinylpyrrolidone composite thermosensitive hydrogels with sustained submucosal lifting for endoscopic submucosal dissection

To develop a submucosal injection material with sustained submucosal lifting for endoscopic submucosal dissection (ESD), this study designed and prepared a novel composite thermosensitive hydrogel system with high pH chitosan-polyvinylpyrrolidone-β-glycerophosphate (HpHCS-PVP-GP). HpHCS improved the injectability of the hydrogels and retained the rapid gelation ability at low concentrations. The modification of PVP significantly improved the stability of low-temperature hydrogel precursor solutions and the integrity of hydrogels formed at 37 °C through hydrogen bonds between PVP and HpHCS. A mathematical model was established using response surface methodology (RSM) to evaluate the synergistic effect of HpHCS, GP, and PVP concentrations on gelation time. This RSM model and submucosal lifting evaluation using in vitro pig esophageal models were used to determine the optimal formula of HpHCS-PVP-GP hydrogels. Although the higher PVP concentration (5 % (w/v)) prolonged gelation time, it improved hydrogel mechanical strength, resulting in better submucosal lifting performance. The experiments of Bama mini pigs showed that the heights of the cushions elevated by the HpHCS-5%PVP-GP hydrogel remained about 80 % 1 h after injection. Repeated injections were avoided, and the hydrogel had no cytotoxicity after electric cutting. Therefore, the HpHCS-PVP-GP thermosensitive hydrogel might be a promising submucosal injection material for ESD.

An ADSC-loaded dermal regeneration template promotes full-thickness wound healing

IntroductionFull-thickness wounds lead to delayed wound healing and scarring. Adipose-derived stem cell (ADSC) grafting promotes wound healing and minimizes scarring, but the low efficiency of grafting has been a challenge. We hypothesized that loading ADSCs onto a clinically widely used dermal regeneration template (DRT) would improve the efficacy of ADSC grafting and promote full-thickness wound healing. MethodsADSCs from human adipose tissue were isolated, expanded, and labeled with a cell tracker. Labeled ADSCs were loaded onto the DRT. The viability, the location of ADSCs on the DRT, and the abundance of ADSCs in the wound area were confirmed using CCK8 and fluorescence microscopy. Full-thickness wounds were created on Bama minipigs, which were applied with sham, ADSC, DRT, and ADSC-DRT. Wounds from the four groups were collected at the indicated time and histological analysis was performed. RNA-seq analysis was also conducted to identify transcriptional differences among the four groups. The identified genes by RNA-seq were verified by qPCR. Immunohistochemistry and western blotting were used to assess collagen deposition. In vitro, the supernatant of ADSCs was used to culture fibroblasts to investigate the effect of ADSCs on fibroblast transformation into myofibroblasts. ResultsADSCs were successfully isolated, marked, and loaded onto the DRT. The abundance of ADSCs in the wound area was significantly greater in the ADSC-DRT group than in the ADSC group. Moreover, the ADSC-DRT group exhibited better wound healing with improved re-epithelialization and denser collagen deposition than the other three groups. The RNA-seq results suggested that the application of the integrated ADSC-DRT system resulted in the differential expression of genes mainly associated with extracellular matrix remodeling. In vivo, wounds from the ADSC-DRT group exhibited an earlier increase in type III collagen deposition and alleviated scar formation. ADSCs inhibited the transformation of fibroblasts into myofibroblasts, along with increased levels of CTGF, FGF, and HGF in the supernatant of ADSCs. Wounds from the ADSC-DRT group had up-regulated expressions of CTGF, HGF, FGF, and MMP3. ConclusionThe integral of ADSC-DRT increased the efficacy of ADSC grafting, and promoted full-thickness wound healing with better extracellular matrix remodeling and alleviated scar formation.

Identification of novel genes associated with atherosclerosis in Bama miniature pig.

Atherosclerosis is a chronic cardiovascular disease of great concern. However, it is difficult to establish a direct connection between conventional small animal models and clinical practice. The pig's genome, physiology, and anatomy reflect human biology better than other laboratory animals, which is crucial for studying the pathogenesis of atherosclerosis. We used whole-genome sequencing data from nine Bama minipigs to perform a genome-wide linkage analysis, and further used bioinformatic tools to filter and identify underlying candidate genes. Candidate gene function prediction was performed using the online prediction tool STRING 12.0. Immunohistochemistry and immunofluorescence were used to detect the expression of proteins encoded by candidate genes. We mapped differential single nucleotide polymorphisms (SNPs) to genes and obtained a total of 102 differential genes, then we used GO and KEGG pathway enrichment analysis to identify four candidate genes, including SLA-1, SLA-2, SLA-3, and TAP2. nsSNPs cause changes in the primary and tertiary structures of SLA-I and TAP2 proteins, the primary structures of these two proteins have undergone amino acid changes, and the tertiary structures also show slight changes. In addition, immunohistochemistry and immunofluorescence results showed that the expression changes of TAP2 protein in coronary arteries showed a trend of increasing from the middle layer to the inner layer. We have identified SLA-I and TAP2 as potential susceptibility genes of atherosclerosis, highlighting the importance of antigen processing and immune response in atherogenesis.

Performance of ATT and UDFF in the diagnosis of non-alcoholic fatty liver: An animal experiment

ObjectiveTo establish a Bama minipigs model with Non-Alcoholic Fatty Liver (NAFL) induced by a high-fat diet and investigate the application of attenuation coefficient (ATT) and ultrasound-derived fat fraction (UDFF) in the diagnosis of NAFL. MethodsSix-month-old male Bama minipigs were randomly divided into normal control and high-fat groups (n = 3 pigs per group), and fed with a control diet and high-fat diet for 32 weeks. Weight and body length were measured every four weeks, followed by quantitative ultrasound imaging (ATT and UDFF), blood biochemical markers, and liver biopsies on the same day. Using the Non-Alcoholic Fatty Liver Disease (NAFLD) Activity Score (NAS) as a reference, we analyzed the correlation between ATT, UDFF, and their score results. ResultsCompared with the normal control group, the body weight, body mass index (BMI), and serum levels of triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in the High-fat group were significantly different at Week 12 (P < 0.05). Spearman correlation analysis showed that the ATT value was significantly correlated with NAS score (r = 0.76, P < 0.001), and the UDFF value was significantly correlated with NAS score (r = 0.80, P < 0.001). The optimal cut-off value of ATT and UDFF were 0.59 dB/cm/MHz and 5.5%, respectively. These values are optimal for diagnosis of NAFL in Bama minipig model. ConclusionATT and UDFF have a high correlation with steatosis, and can be used as a non-invasive method for early screening of hepatic steatosis, which can dynamically monitor the change of disease course.

MX1 and UBE2L6 are potential metaflammation gene targets in both diabetes and atherosclerosis.

The coexistence of diabetes mellitus (DM) and atherosclerosis (AS) is widespread, although the explicit metabolism and metabolism-associated molecular patterns (MAMPs) responsible for the correlation are still unclear. Twenty-four genetically wild-type male Ba-Ma mini pigs were randomly divided into five groups distinguished by different combinations of 90 mg/kg streptozotocin (STZ) intravenous injection and high-cholesterol/lipid (HC) or high-lipid (HL) diet feeding for 9 months in total. Pigs in the STZ+HC and STZ+HL groups were injected with STZ first and then fed the HC or HL diet for 9 months. In contrast, pigs in the HC+STZ and HL+STZ groups were fed the HC or HL diet for 9 months and injected with STZ at 3 months. The controls were only fed a regular diet for 9 months. The blood glucose and abdominal aortic plaque observed through oil red O staining were used as evaluation indicators for successful modelling of DM and AS. A microarray gene expression analysis of all subjects was performed. Atherosclerotic lesions were observed only in the HC+STZ and STZ+HC groups. A total of 103 differentially expressed genes (DEGs) were identified as common between them. The most significantly enriched pathways of 103 common DEGs were influenza A, hepatitis C, and measles. The global and internal protein-protein interaction (PPI) networks of the 103 common DEGs consisted of 648 and 14 nodes, respectively. The top 10 hub proteins, namely, ISG15, IRG6, IRF7, IFIT3, MX1, UBE2L6, DDX58, IFIT2, USP18, and IFI44L, drive aspects of DM and AS. MX1 and UBE2L6 were the intersection of internal and global PPI networks. The expression of MX1 and UBE2L6 was 507.22 ± 342.56 and 96.99 ± 49.92 in the HC+STZ group, respectively, which was significantly higher than others and may be linked to the severity of hyperglycaemia-related atherosclerosis. Further PPI network analysis of calcium/micronutrients, including MX1 and UBE2L6, consisted of 58 and 18 nodes, respectively. The most significantly enriched KEGG pathways were glutathione metabolism, pyrimidine metabolism, purine metabolism, and metabolic pathways. The global and internal PPI network of the 103 common DEGs consisted of 648 and 14 nodes, respectively. The intersection of the nodes of internal and global PPI networks was MX1 and UBE2L6, suggesting their key role in the comorbidity mechanism of DM and AS. This inference was partly verified by the overexpression of MX1 and UBE2L6 in the HC+STZ group but not others. Further calcium- and micronutrient-related enriched KEGG pathway analysis supported that MX1 and UBE2L6 may affect the inflammatory response through micronutrient metabolic pathways, conceptually named metaflammation. Collectively, MX1 and UBE2L6 may be potential common biomarkers for DM and AS that may reveal metaflammatory aspects of the pathological process, although proper validation is still needed to determine their contribution to the detailed mechanism.

Enhanced Airway Hyperresponsiveness by Up-Regulation of Tumor Necrosis Factor Αlpha Pathway in Long-Term High-Fat and Sucrose Diet in Bama Mini Pigs

Enhanced Airway Hyperresponsiveness by Up-Regulation of Tumor Necrosis Factor Αlpha Pathway in Long-Term High-Fat and Sucrose Diet in Bama Mini Pigs

Flavonoids from mulberry leaves inhibit fat production and improve fatty acid distribution in adipose tissue in finishing pigs

This study evaluated the effects of flavonoids from mulberry leaves (FML) on plasma biochemical indices, serum activities of lipid metabolism-related enzymes, fat morphology, fatty acid composition, and lipid metabolism in different adipose tissues of finishing pigs. We used 120 Chinese hybrid barrows of Berkshire and Bama mini-pigs with an average initial body weight of 45.11 ± 4.23 kg. The pigs were randomly assigned to five treatment groups and fed a control diet based on corn, soybean meal, and wheat bran or a control diet supplemented with 0.02%, 0.04%, 0.08%, or 0.16% FML. Each experimental group had six replicates (pens), with four pigs per pen. After a 7-d adaptation period, the feeding trial was conducted for 58 d. Blood and adipose tissue samples were collected from 30 pigs (one pig per pen) at the end of the test. The results showed that FML supplementation significantly decreased the feed intake to body gain ratio, the plasma concentrations of total cholesterol and free fatty acids, and the serum activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (linear or quadratic effects, P < 0.05), and decreased the plasma triglyceride concentration (quadratic, P = 0.07). Increasing FML supplementation increased the average daily gain and serum activities of lipoprotein lipase (linear and quadratic effects, P < 0.05) and adipose triglyceride lipase (linear, P < 0.05). Dietary FML supplementation decreased the adipocyte area in the dorsal subcutaneous adipose (DSA) tissue of finishing pigs (linear, P = 0.05) and increased the adipocyte area in the visceral adipose tissue (quadratic, P < 0.01). Increasing FML supplementation decreased the C20:1 content in DSA, abdominal subcutaneous adipose, and visceral adipose tissues of finishing pigs (P < 0.05) and increased the C18:3n3 and n-3 PUFA contents (P < 0.05). The lipid metabolism genes were regulated by the PPARγ-LXRα-ABCA1 signaling pathway, and their expressions differed in different adipose tissues. These findings suggest that FML improved growth performance, regulated lipid metabolism, inhibited fat production, and improved fatty acid distribution in the adipose tissue of finishing pigs, thereby improving pig fat's nutritional quality and health value.

Dietary Betaine Supplementation Enhances Colonic Barrier Function through the Nrf2/Keap1 and TLR4-NF-κB/MAPK Signaling Pathways and Alters Colonic Microbiota in Bama Mini-Pigs.

This study evaluated the effects of betaine supplementation in sows and/or their offspring's diets on the redox status, immune and inflammatory levels, colonic barrier function, and colonic microbial community of offspring piglets. Thirty-six Bama mini-sows on day 3 of gestation and their weaned offspring piglets (28 d of age) were randomly allocated to the following treatments: (1) sows and their weaned offspring fed the basal diet (control group, Con group); (2) sows fed the basal diet with 3.50 kg/t betaine, and their weaned offspring fed the basal diet (sows betaine group, SB group); (3) sows fed the basal diet with 3.50 kg/t betaine, and their weaned offspring fed the basal diet with 2.50 kg/t betaine (sow-offspring betaine group, S-OB group). Six offspring piglets from each group were selected to collect plasma and colon samples on d 30, 60, and 90 after weaning. Compared with the Con group, the plasma levels of IgA, IgM, GSH-Px, and SOD during d 30-90 after weaning, IFN-α, T-AOC, and GSH on d 30 and 60 after weaning were increased, while MDA during d 30-90 after weaning was decreased in the SB and S-OB groups (p < 0.05). In addition, the plasma levels of IFN-γ on d 60 and T-AOC on d 30 after weaning were higher in the S-OB group than those in the Con group (p < 0.05). In the colon, betaine supplementation increased plasma T-AOC, GSH, and SOD levels while decreasing MDA concentration (p < 0.05). Betaine supplementation improved the colonic protein abundances of ZO-1, occludin, and claudin in offspring and activated the Nrf2/Keap1 signaling pathway while inhibiting the TLR4-NF-κB/MAPK signaling pathway on d 90 after weaning. The 16S rRNA sequencing results showed that betaine supplementation altered colonic microbiota composition by increasing the relative abundances of Verrucomicrobia and Actinobacteria in the SB group while decreasing proinflammatory-associated microbiota abundances (Tenericutes, Prevotella, and Parabacteroides) (p < 0.05). Collectively, these findings suggest that dietary betaine supplementation in sows and/or their offspring could improve offspring piglets' redox status and immune and anti-inflammatory levels and enhance the colonic barrier function by activating Nrf2/Keap1 and inhibiting TLR4-NF-κB/MAPK signaling pathways.

Dynamic changes of fecal microbiota in a weight-change model of Bama minipigs.

Obesity is closely related to gut microbiota, however, the dynamic change of microbial diversity and composition during the occurrence and development process of obesity is not clear. A weight-change model of adult Bama pig (2 years, 58 individuals) was established, and weight gain (27 weeks) and weight loss (9 weeks) treatments were implemented. The diversity and community structures of fecal microbiota (418 samples) was investigated by using 16S rRNA (V3-V4) high-throughput sequencing. During the weight gain period (1~27 week), the alpha diversity of fecal microbiota exhibited a "down-up-down" fluctuations, initially decreasing, recovering in the mid-term, and decreasing again in the later stage. Beta diversity also significantly changed over time, indicating a gradual deviation of the microbiota composition from the initial time point. Bacteroides, Clostridium sensu stricto 1, and Escherichia-Shigella showed positive correlations with weight gain, while Streptococcus, Oscillospira, and Prevotellaceae UCG-001 exhibited negative correlations. In the weight loss period (30~38 week), the alpha diversity further decreased, and the composition structure underwent significant changes compared to the weight gain period. Christensenellaceae R-7 group demonstrated a significant increase during weight loss and showed a negative correlation with body weight. Porphyromonas and Campylobacter were positively correlated with weight loss. Both long-term fattening and weight loss induced by starvation led to substantial alterations in porcine gut microbiota, and the microbiota changes observed during weight gain could not be recovered during weight loss. This work provides valuable resources for both obesity-related research of human and microbiota of pigs.

P24-01: A Review of the Incidence of Common Arrhythmias in Bama Minipigs observed in Stand-alone Safety Pharmacology and General Toxicology Studies

P24-01: A Review of the Incidence of Common Arrhythmias in Bama Minipigs observed in Stand-alone Safety Pharmacology and General Toxicology Studies

Dermal repeated dose toxicity study of the anti-breast cancer drug Formestane cream in Bama minipig.

Formestane (4-OHA) has been proven to be highly effective with high systemic tolerability in treating ER+ breast cancer. However, its intramuscular administration and associated side effects make it unsuitable for adjuvant treatment, leading to its withdrawal from the market. In contrast, Formestane cream may offer a solution by providing a more convenient route of administration and retaining its tumor-shrinking effects. This suggests that 4-OHA cream could have promising clinical applications. However, before clinical application, it is necessary to evaluate the potential toxicity of the cream in animals. This study evaluated the toxicity of 4-OHA cream on female Bama minipigs in vivo by analyzing hematology, biochemistry, and histopathology. The results showed that there was no significant difference between the cream-treated group and the control normal group for each parameter analyzed, indicating that 4-OHA cream was non-dermal toxic to minipigs. This finding provides a basis for the safe clinical use of the cream.

A LC-MS/MS method for determination of clenbuterol enantiomers in animal tissues and its application to the enantioselective distribution in Bama mini-pigs

ObjectivesTo establish and validate a simple and reliable analytical method for separation and determination of clenbuterol enantiomers (R-(-)-clenbuterol &S-(+)-clenbuterol) in animal tissues, and apply it to the enantioselective distribution of clenbuterol in Bama mini-pigs. MethodsA LC-MS/MS analytical method was developed and validated in positive multiple reaction monitoring mode with electrospray ionization. After perchloric acid deproteinization, samples were pretreated only by one step liquid–liquid extraction using tert-butyl methyl ether under strong alkaline condition. Teicoplanin was used as chiral selector and 10 mM ammonium formate methanol solution was used as mobile phase. The optimized chromatographic separation conditions were completed in 8 min. Two chiral isomers in 11 edible tissues from Bama mini-pigs were investigated. ResultsR-(-)-clenbuterol and S-(+)-clenbuterol can be baseline separated and accurately analyzed with a linear range of 5–500 ng/g. Accuracies ranged from −11.9–13.0% for R-(-)-clenbuterol and −10.2–13.2% for S-(+)-clenbuterol, intra-day and inter-day precisions were between 0.7 and 6.1% for R-(-)-clenbuterol and 1.6–5.9% for S-(+)-clenbuterol. R/S ratios in edible tissues of pigs were all significantly lower than 1. ConclusionsThe analytical method has good specificity and robustness in determination of R-(-)-clenbuterol and S-(+)-clenbuterol in animal tissues, and can be used as a routine analysis method for food safety and doping control. There is a significant difference in R/S ratio between pig feeding tissues and pharmaceutical preparations (racemate with R/S ratio of 1), which makes it possible to identify the source of clenbuterol in doping control and investigation.

Isolation of porcine adult cardiomyocytes: Comparison between Langendorff perfusion and tissue slicing-assisted enzyme digestion.

Tissue slicing-assisted digestion (TSAD) of adult cardiomyocytes has shown significant improvements over conventional chunk methods. However, it remains unclear how this method compares to Langendorff perfusion, the current standard of adult cardiomyocyte isolation. Using adult Bama minipigs, we performed cardiomyocyte isolation via these two distinct methods, and compared the resulting cellular quality, including viability, cellular structure, gene expression, and electrophysiological properties, of cardiomyocytes from 3 distinct anatomical regions, namely the left ventricle, right ventricle, and left atrial appendage. Our results revealed largely indistinguishable cell quality in all of the measured parameters. These findings suggest that that TSAD can be reliably used to isolate adult mammalian cardiomyocytes as a reliable alternative to perfusion in cardiomyocyte isolation from larger mammals, particularly when Langendorff perfusion is not feasible.

Whole-genome sequencing study to identify candidate markers indicating susceptibility to type 2 diabetes in Bama miniature pigs.

Hundreds of single-nucleotide polymorphism (SNP) sites have been found to be potential genetic markers of type 2 diabetes mellitus (T2DM). However, SNPs related to T2DM in minipigs have been less reported. This study aimed to screen the T2DM-susceptible candidate SNP loci in Bama minipigs so as to improve the success rate of the minipig T2DM model. The genomic DNAs of three Bama minipigs with T2DM, six sibling low-susceptibility minipigs with T2DM, and three normal control minipigs were compared by whole-genome sequencing. The T2DM Bama minipig-specific loci were obtained, and their functions were annotated. Meanwhile, the Biomart software was used to perform homology alignment with T2DM-related loci obtained from the human genome-wide association study to screen candidate SNP markers for T2DM in Bama miniature pigs. Whole-genome resequencing detected 6960 specific loci in the minipigs with T2DM, and 13 loci corresponding to 9 diabetes-related genes were selected. Further, a set of 122 specific loci in 69 orthologous genes of human T2DM candidate genes were obtained in the pigs. Collectively, a batch of T2DM-susceptible candidate SNP markers in Bama minipigs, covering 16 genes and 135 loci, was established. Whole-genome sequencing and comparative genomics analysis of the orthologous genes in pigs that corresponded to the human T2DM-related variant loci successfully screened out T2DM-susceptible candidate markers in Bama miniature pigs. Using these loci to predict the susceptibility of the pigs before constructing an animal model of T2DM may help to establish an ideal animal model.

Sow-Offspring Diets Supplemented with Probiotics and Synbiotics Are Associated with Offspring's Growth Performance and Meat Quality.

Probiotics and synbiotics supplementation have been shown to play potential roles in animal production. The present study aimed to evaluate the effects of dietary probiotics and synbiotics supplementation to sows during gestation and lactation and to offspring pigs (sow-offspring) on offspring pigs' growth performance and meat quality. Sixty-four healthy Bama mini-pigs were selected and randomly allocated into four groups after mating: the control, antibiotics, probiotics, and synbiotics groups. After weaning, two offspring pigs per litter were selected, and four offspring pigs from two litters were merged into one pen. The offspring pigs were fed a basal diet and the same feed additive according to their corresponding sows, representing the control group (Con group), sow-offspring antibiotics group (S-OA group), sow-offspring probiotics group (S-OP group), and sow-offspring synbiotics group (S-OS group). Eight pigs per group were euthanized and sampled at 65, 95, and 125 d old for further analyses. Our findings showed that probiotics supplementation in sow-offspring diets promoted growth and feed intake of offspring pigs during 95-125 d old. Moreover, sow-offspring diets supplemented with probiotics and synbiotics altered meat quality (meat color, pH45min, pH24h, drip loss, cooking yield, and shear force), plasma UN and AMM levels, and gene expressions associated with muscle-fiber types (MyHCI, MyHCIIa, MyHCIIx, and MyHCIIb) and muscle growth and development (Myf5, Myf6, MyoD, and MyoG). This study provides a theoretical basis for the maternal-offspring integration regulation of meat quality by dietary probiotics and synbiotics supplementation.