Dietary probiotic and synbiotic supplementation starting from maternal gestation improves muscular lipid metabolism in offspring piglets by reshaping colonic microbiota and metabolites.

Probiotics and synbiotics have been intensively used in animal husbandry due to their advantageous roles in animals' health. However, there is a paucity of research on probiotic and synbiotic supplementation from maternal gestation to the postnatal growing phases of offspring piglets. Thus, we assessed the effects of dietary supplementation of these two additives to sows and offspring piglets on skeletal muscle and body metabolism, colonic microbiota composition, and metabolite profiles of offspring piglets. Pregnant Bama mini-pigs and their offspring piglets (after weaning) were fed either a basal diet or a basal diet supplemented with antibiotics, probiotics, or synbiotics. At 65, 95, and 125 days old, eight pigs per group were euthanized and sampled for analyses. Probiotics increased the intramuscular fat content in the psoas major muscle (PMM) at 95 days old, polyunsaturated fatty acid (PUFA) and n-3 PUFA levels in the longissimus dorsi muscle (LDM) at 65 days old, C16:1 level in the LDM at 125 days old, and upregulated ATGL, CPT-1, and HSL expressions in the PMM at 65 days old. Synbiotics increased the plasma HDL-C level at 65 days old and TC level at 65 and 125 days old and upregulated the CPT-1 expression in the PMM at 125 days old. In addition, probiotics and synbiotics increased the plasma levels of HDL-C at 65 days old, CHE at 95 days old, and LDL-C at 125 days old, while decreasing the C18:1n9t level in the PMM at 65 days old and the plasma levels of GLU, LDH, and TG at 95 days old. Microbiome analysis showed that probiotic and synbiotic supplementation increased colonic Actinobacteria, Firmicutes, Verrucomicrobia, Faecalibacterium, Pseudobutyrivibrio, and Turicibacter abundances. However, antibiotic supplementation decreased colonic Actinobacteria, Bacteroidetes, Prevotella, and Unclassified_Lachnospiraceae abundances. Furthermore, probiotic and synbiotic supplementation was associated with alterations in 8, 7, and 10 differential metabolites at three different age stages. Both microbiome and metabolome analyses showed that the differential metabolic pathways were associated with carbohydrate, amino acid, and lipid metabolism. However, antibiotic supplementation increased the C18:1n9t level in the PMM at 65 days old and xenobiotic biodegradation and metabolism at 125 days old. In conclusion, sow-offspring's diets supplemented with these two additives showed conducive effects on meat flavor, nutritional composition of skeletal muscles, and body metabolism, which may be associated with the reshaping of colonic microbiota and metabolites. However, antibiotic supplementation has negative effects on colonic microbiota composition and fatty acid composition in the PMM. IMPORTANCE: The integral sow-offspring probiotic and synbiotic supplementation improves the meat flavor and the fatty acid composition of the LDM to some extent. Sow-offspring probiotic and synbiotic supplementation increases the colonic beneficial bacteria (including Firmicutes, Verrucomicrobia, Actinobacteria, Faecalibacterium, Turicibacter, and Pseudobutyrivibrio) and alters the colonic metabolite profiles, such as guanidoacetic acid, beta-sitosterol, inosine, cellobiose, indole, and polyamine. Antibiotic supplementation in sow-offspring's diets decreases several beneficial bacteria (including Bacteroidetes, Actinobacteria, Unclassified_Lachnospiraceae, and Prevotella) and has a favorable effect on improving the fatty acid composition of the LDM to some extent, while presenting the opposite effect on the PMM.

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.

Brincidofovir is a robust replication inhibitor against African swine fever virus in vivo and in vitro.

African swine fever virus (ASFV) infection is a major public and socioeconomic concern that has a serious impact on the global swine industry. Unfortunately, there are currently no commercially available vaccines or antiviral agents that are both safe and effective against ASFV. In the study, we use primary porcine alveolar macrophages to screen a kinase inhibitor library for anti-ASFV compounds. Six candidate compounds that inhibited ASFV infection with inhibition of > 90% were identified, among which brincidofovir exhibited optimal inhibitory effects on ASFV. Brincidofovir reduces ASFV replication in a dose-dependent manner (IC50 = 2.76 nM) without cytotoxicity (CC50 = 58 µM). It possesses the ability to reduce viral titres and inhibit viral structural protein expression. Time-of-addition assays suggest that the compound interferes with the post-invasion stage of the viral infection cycle. In pig challenge experiments, brincidofovir was indicated to protect pigs against ASFV-induced lethality by decreasing the viral load in organs and peripheral blood, while it alleviated the histopathological changes associated with ASFV infection. Furthermore, brincidofovir also decreased viral shedding in pigs with ASFV infection. Our data together demonstrate that brincidofovir may serve as a potentially effective agent for the prevention and control of ASFV infection, whereas further investigations are still required.

Infection Characteristics and Transcriptomics of African Swine Fever Virus in Bama Minipigs.

Animal experiments on African swine fever virus (ASFV) are vital to the study of ASFV; however, ASFV can only infect pigs, and animal experiments need to be performed in animal biosafety level 3 (ABSL-3) laboratories, meaning that many small ABSL-3 laboratories are unable to carry out in vivo ASFV experiments. Therefore, miniaturized experimental animals for ASFV infection are urgently needed. Here, we successfully isolated genotype II of ASFV SY-1 from wild boars and evaluated ASFV-infected Bama minipigs in a negative-pressure isolator of a small ABSL-3 laboratory. The pathological changes of ASFV-infected Bama minipigs were consistent with characteristic lesions of ASFV-infected domestic pigs and wild boars. All pigs died 5 to 14 days postinfection (dpi) through intramuscular injection. Viral genomic DNA from nasal, oral, and rectal swab samples was first detectable at 2 to 4 dpi. The common differentially expressed genes were clustered in the immune-related, metabolic, and inflammatory response pathways from the spleen and inguinal lymph node samples comparing infected to mock. In summary, these results demonstrated that the Bama minipig was an appropriate model for ASFV infection in small ABSL-3 laboratories that can accelerate the research of vaccines and antiviral drugs and uncover pathogenic mechanisms of ASFV infection. IMPORTANCE African swine fever virus (ASFV) can only infect pigs rather than other animals. However, the domestic pigs cannot be kept in small ABSL-3 laboratories for a long time due to the characteristics of rapid growth and large size, which hinder ASFV research, including research of vaccines, antiviral drugs, and mechanisms. In contrast, Bama minipigs have unique advantages consisting of low growth and small size. In the research, Bama minipigs were used to evaluate the characteristics of ASFV infection in small ABSL-3 laboratories. The pathological changes, viral shedding, and gene regulation were consistent with those of domestic pigs infected with ASFV. Therefore, Bama minipigs can be a suitable model for ASFV infection in small ABSL-3 laboratories.

Maternal probiotics supplementation improves immune and antioxidant function in suckling piglets via modifying gut microbiota.

AIM: Probiotics could improve the health, growth, and development of host or their foetuses/offspring via regulating gut microbiota. The present study was conducted to determine the effects of maternal probiotics supplementation on gut microbiota and metabolites of sows and their suckling piglets, as well as plasma biochemical parameters, oxidative/anti-oxidative indexes, and inflammatory cytokine levels of suckling piglets. METHODS AND RESULTS: A total of 32 pregnant Bama mini-pigs were selected and randomly divided into two groups. The sows were fed a basal diet (control group) or a basal diet supplemented with probiotics (probiotics group) from mating to day 21 of lactation. Samples from sows were collected on day 105 of pregnancy and day 21 of lactation and from piglets on day 21 of lactation. The results showed that probiotics supplementation increased the faecal abundances of Ruminococcus, Bacteroides, and Anaeroplasma and decreased Tenericutes on day 105 of pregnancy while increased the abundances of Actinobacteria and Anaerostipes and decreased Proteobacteria and Desulfovibrio on day 21 of lactation. In addition, probiotics supplementation decreased the faecal levels of tryptamine, putrescine, and cadaverine on day 105 of pregnancy and isovalerate and skatole on day 21 of lactation while increased butyrate level on day 21 of lactation. Further studies showed that maternal probiotics supplementation decreased the plasma levels of AMM, TC, LDL-C, Ala, Tau, MDA, H2 O2 , IL-1ß, IL-2, IL-6, and IFN-α of suckling piglets. Moreover, maternal probiotics supplementation increased the abundances of Deferribacteres, Fusobacteria, and Fusobacterium while decreased Anaerostipes in piglet's colon. Spearman's correlation analysis revealed a potential link between gut microbiota alterations and their metabolites. CONCLUSIONS: Dietary probiotics supplementation during pregnancy and lactation periods could improve sow status, alleviate oxidative stress and inflammation response, and improve nutrient metabolism of piglets by altering the gut microbiota. SIGNIFICANCE AND IMPACT OF THE STUDY: The probiotics alter maternal and offspring's gut microbiota involving in offspring's physiological and metabolic changes, and present a new perspective that the effects of gut microbiota changes induced by probiotics supplementation will help in addressing the growth and development and health problem of their foetuses/offspring.

Dietary Betaine Addition Alters Carcass Traits, Meat Quality, and Nitrogen Metabolism of Bama Mini-Pigs.

Betaine is widely used as feed additives in animal husbandry as it can cause many benefits such as improving antioxidant ability, growth performance, and carcass traits. However, there are limited studies about the effects of betaine on the Bama mini-pigs. The present study was conducted to evaluate the effects of dietary betaine on carcass traits, meat quality, and nitrogen metabolism of pigs. Twenty-six pregnant Bama mini-pigs and then 104 weaned piglets were assigned for experimental treatments. The plasma and muscle samples were collected at 65-, 95-, and 125-d-old pigs, respectively. The results showed that betaine addition in the sow-offspring diets increased the lean meat rate in the 65-d-old pigs, whereas carcass weight, carcass yield, and loin-eye area were increased in the 95-d-old pigs, and carcass weight and backfat thickness in the 125-d-old pigs. Dietary betaine addition in the sow-offspring diets increased the contents of plasma Asp of 65-d-old, Met of 95- and 125-d-old, and Sar of 125-d-old pigs. Moreover, betaine addition increased the contents of Met, His, Ile, and Phe in Longissimus thoracis et lumborum, whereas those contents were decreased in biceps femoris and psoas major muscles at different stages. Betaine addition in the sow and piglets' diets regulated the muscle fiber-type and myogenic regulatory gene expressions. In summary, betaine addition in the sow and sow-offspring diets could improve the carcass traits and meat quality by altering the plasma biochemical parameters, amino acid composition, and gene expressions of skeletal muscle.

Application of the modified cytosine base-editing in the cultured cells of bama minipig.

Bama minipig is a unique miniature swine bred from China. Their favorable characteristics include delicious meat, strong adaptability, tolerance to rough feed, and high levels of stress tolerance. Unfavorable characteristics are their low lean meat percentage, high fat content, slow growth rate, and low feed conversion ratio. Genome-editing technology using CRISPR/Cas9 efficiently knocked out the myostatin gene (MSTN) that has a negative regulatory effect on muscle production, effectively promoting pig muscle growth and increasing lean meat percentage of the pigs. However, CRISPR/Cas9 genome editing technology is based on random mutations implemented by DNA double-strand breaks, which may trigger genomic off-target effects and chromosomal rearrangements. The application of CRISPR/Cas9 to improve economic traits in pigs has raised biosafety concerns. Base editor (BE) developed based on CRISPR/Cas9 such as cytosine base editor (CBE) effectively achieve targeted modification of a single base without relying on DNA double-strand breaks. Hence, the method has greater safety in the genetic improvement of pigs. The aim of the present study is to utilize a modified CBE to generate MSTN-knockout cells of Bama minipigs. Our results showed that the constructed "all-in-one"-modified CBE plasmid achieved directional conversion of a single C·G base pair to a T·A base pair of the MSTN target in Bama miniature pig fibroblast cells. We successfully constructed multiple single-cell colonies of Bama minipigs fibroblast cells carrying the MSTN premature termination and verified that there were no genomic off-target effects detected. This study provides a foundation for further application of somatic cell cloning to construct MSTN-edited Bama minipigs that carry only a single-base mutation and avoids biosafety risks to a large extent, thereby providing experience and a reference for the base editing of other genetic loci in Bama minipigs.

A guangxi bama minipig model of trimeresurus stejnegeri snakebite: Modeling and evaluation / 中国组织工程研究

BACKGROUND: At present, there are not many research methods on the pathological mechanism of Trimeresurus stejnegeri snakebite, and there are few methods for constructing animal models. OBJECTIVE: To establish and evaluate the Guangxi Bama minipig model for research on the pathological mechanism, diagnosis and treatment of Trimeresurus stejnegeri snakebite. METHODS: Based on the 50% lethal dose of intramuscularly injected Trimeresurus stejnegeri venom to mice, the theoretical 50% lethal venom dose for Bama minipigs was calculated by the equivalent dose coefficient conversion and reduction algorithm, and the body surface area conversion algorithm. Twelve Bama minipigs were randomly divided into a normal group (n=6) and a model group (n=6). The model group was injected intramuscularly with 0.2 mL/kg snake venom 1/3 of the theoretical 50% lethal dose (0.643 mg/kg). The control group was injected with the same amount of normal saline. After snake venom injection, the poisoning symptoms of Bama minipigs were observed. Two groups of animal blood samples were collected before, 6 hours and 24 hours after snake venom injection. Blood routine test, four coagulation items, blood biochemistry and electrolyte were detected. Histopathological changes of the heart, brain, lung, liver, and kidney as well as the injection site were observed by hematoxylin-eosin staining. The study protocol was approved by the Laboratory Animal Ethics Committee (approval No. 201909013). RESULTS AND CONCLUSION: There was swelling at the wound of the piglet accompanied with blood blisters after snake venom injection. The affected limbs and the surrounding area were swollen and spread rapidly to the proximal end. The piglets walked all the time because of the pain, and no animal died during the experiment. Compared with the control group pig, the model group had higher red blood cell count, white blood cell count, alanine aminotransferase, D-dimer count, longer prothrombin time, and lower fibrinogen and platelet count. Hematoxylin-eosin staining showed that, compared with the control group, capillary permeability of piglet lung tissues in the model group was increased with hyperemia and edema. Edema, bleeding, degeneration and necrosis were seen in the muscle tissue on the injection site. No obvious abnormalities in other organs and tissues were observed. To conclude, this method can be used to establish a pig model of Trimeresurus stejnegeri snakebite that can reflect the pathophysiological process of Trimeresurus stejnegeri snakebite. It has operability and repeatability that can be used to study the pathophysiological mechanism of Trimeresurus stejnegeri snakebite.

CRISPR/Cas9-Mediated Biallelic Knockout of IRX3 Reduces the Production and Survival of Somatic Cell-Cloned Bama Minipigs.

Bama minipigs are a local pig breed that is unique to China and has a high development and utilization value. However, its high fat content, low feed utilization rate, and slow growth rate have limited its popularity and utilization. Compared with the long breeding cycle and high cost of traditional genetic breeding of pigs, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) endonuclease 9 system (CRISPR/Cas9)-mediated gene editing can cost-effectively implement targeted mutations in animal genomes, thereby providing a powerful tool for rapid improvement of the economic traits of Bama minipigs. The iroquois homeobox 3 (IRX3) gene has been implicated in human obesity. Mouse experiments have shown that knocking out IRX3 significantly enhances basal metabolism, reduces fat content, and controls body mass and composition. This study aimed to knock out IRX3 using the CRISPR/Cas9 gene editing method to breed Bama minipigs with significantly reduced fat content. First, the CRISPR/Cas9 gene editing method was used to efficiently obtain IRX3-/- cells. Then, the gene-edited cells were used as donor cells to produce surviving IRX3-/- Bama minipigs using somatic cell cloning. The results show that the use of IRX3-/- cells as donor cells for the production of somatic cell-cloned pigs results in a significant decrease in the average live litter size and a significant increase in the average number of stillbirths. Moreover, the birth weight of surviving IRX3-/- somatic cell-cloned pigs is significantly lower, and viability is poor such that all piglets die shortly after birth. Therefore, the preliminary results of this study suggest that IRX3 may have important biological functions in pigs, and IRX3 should not be used as a gene editing target to reduce fat content in Bama minipigs. Moreover, this study shows that knocking out IRX3 does not favor the survival of pigs, and whether targeted regulation of IRX3 in the treatment of human obesity will also induce severe adverse consequences requires further investigation.

Naoxintong Capsule Inhibits the Development of Cardiovascular Pathological Changes in Bama Minipig Through Improving Gut Microbiota.

Naoxintong capsule (NXT), a Chinese medicine, has performed excellent effects on the prevention and treatment against cardiovascular diseases. NXT is a fine powder mixture without any herb extraction, and there must be lots of ingredients hard to be absorbed. However, little is known about the correlation between the NXT's cardioprotective effects and gut microbiota. Herein, we report the effect of NXT on the development of cardiovascular diseases and clarify the correlation between NXT's cardioprotective effects and gut microbiota. In the current study, minipigs were selected and fed with high-fat diet and NXT daily for successive 8 months. During the process, up to 18 biomedical parameters were monthly determined to observe the dynamic changes after NXT treatment. At the end of experimental process, pathological examinations of heart, coronary artery, carotid artery, thoracic aorta, and abdominal aorta were conducted by HE staining and 16SrDNA sequencing, and analyzing of gut microbiota were conducted. Our results showed that NXT's effects against cardiovascular diseases were through regulating blood lipid profiles, inhibiting vascular inflammation, enhancing antioxidant capacity, and alleviating myocardial injury, without damages on liver and kidney particularly. Concurrently, we also found that long-term administration of NXT increased the diversity of gut microbiota, influenced the microbiome structure and composition stably, and revered the increase of the ratio of the Firmicutes to Bacteroidetes (F/B ratio) in relative abundance. Specifically, our results revealed some key bacterium of Caproiciproducens (enhanced), Sutterella (enhanced), Erysipelotrichaceae (enhanced), and Romboutsia (decreased) that were closely involved in NXT's effects. Taken together, our study demonstrates that NXT can inhibit the development of cardiovascular diseases by ameliorating high-fat diet-induced metabolic disorders and partly through improving gut microbiota.

Efficient generation of GHR knockout Bama minipig fibroblast cells using CRISPR/Cas9-mediated gene editing.

Dwarfism, also known as growth hormone deficiency (GHD), is a disease caused by genetic mutations that result in either a lack of growth hormone or insufficient secretion of growth hormone, resulting in a person's inability to grow normally. In the past, many studies focusing on GHD have made use of models of other diseases such as metabolic or infectious diseases. A viable GHD specific model system has not been used previously, thus limiting the interpretation of GHD results. The Bama minipig is unique to Guangxi province and has strong adaptability and disease resistance, and an incredibly short stature, which is especially important for the study of GHD. In addition, studies of GHR knockout Bama minipigs and GHR knockout Bama minipig fibroblast cells generated using CRISPR/Cas9 have not been previously reported. Therefore, the Bama minipig was selected as an animal model and as a tool for the study of GHD in this work. In this study, a Cas9 plasmid with sgRNA targeting the first exon of the GHR gene was transfected into Bama minipig kidney fibroblast cells to generate 22 GHR knockout Bama minipig kidney fibroblast cell lines (12 male monoclonal cells and 10 female monoclonal cells). After culture and identification, 11 of the 12 male clone cell lines showed double allele mutations, and the rate of positive alteration of GHR was 91.67%. Diallelic mutation of the target sequence occurred in 10 female clonal cell lines, with an effective positive mutation rate of 100%. Our experimental results not only showed that CRISPR/Cas9 could efficiently be used for gene editing in Bama minipig cells but also identified a highly efficient target site for the generation of a GHR knockout in other porcine models. Thus, the generation of GHR knockout male and female Bama fibroblast cells could lay a foundation for the birth of a future dwarfism model pig. We anticipate that the "mini" Bama minipig will be of improved use for biomedical and agricultural scientific research and for furthering our understanding of the genetic underpinnings of GHD.

Transcriptome Level Changes in Animal Model of Scars During Wound Healing and Pressure Therapy / 医用生物力学

Objective To study the changes of transcriptome levels in a Bama minipig model of hypertrophic scar during wound healing and pressure therapy by using RNA sequencing (RNA-seq) technique. Methods The Bama minipig model was established by skin wounds from the back and pressure (3.4 kPa) was initiated at 60 days after skin injury. Total RNA was extracted from scar tissues at 0, 14, 30, 60, and 90 days after skin injury and then sequenced. The resulting sequences were mapped to porcine reference genomes and transcriptomes were reconstructed to search for differentially expressed genes (DEGs). The DEGs were further subjected to GO and KEGG analysis using bioinformatics method, while part of the genes were selected for verification using qRT-PCR. Results After preprocessing, more than 78% reads in each group were accurately aligned to the reference sequence. The DEGs identification result showed that 568 genes were differentially expressed after pressure treatment, with 289 up-regulated and 279 down-regulated. GO enrichment analysis revealed that DEGs in each group were mainly associated with extracellular matrix, tissue development and skin development. KEGG analysis showed that the DEGs in each group during wound healing were mainly enriched in extracellular matrix-receptor interactions, focal adhesion and apoptosis pathways; while the DEGs after pressure treatment were mainly enriched in PI3K-Akt and MAPK signaling pathway except the pathways mentioned. qRT-PCR showed that the expression patterns of 6 DEGs were consistent with RNA-seq analysis, confirming the reliability of RNA-seq result. Conclusions RNA-Seq analysis identified differentially expressed genes in animal model of scars during wound healing and pressure therapy, which provided experimental evidence for clinical scar treatment.

Transcriptome Level Changes in Animal Model of Scars During Wound Healing and Pressure Therapy / 医用生物力学

Objective To study the changes of transcriptome levels in a Bama minipig model of hypertrophic scar during wound healing and pressure therapy by using RNA sequencing (RNA-seq) technique. Methods The Bama minipig model was established by skin wounds from the back and pressure (3.4 kPa) was initiated at 60 days after skin injury. Total RNA was extracted from scar tissues at 0, 14, 30, 60, and 90 days after skin injury and then sequenced. The resulting sequences were mapped to porcine reference genomes and transcriptomes were reconstructed to search for differentially expressed genes (DEGs). The DEGs were further subjected to GO and KEGG analysis using bioinformatics method, while part of the genes were selected for verification using qRT-PCR. Results After preprocessing, more than 78% reads in each group were accurately aligned to the reference sequence. The DEGs identification result showed that 568 genes were differentially expressed after pressure treatment, with 289 up-regulated and 279 down-regulated. GO enrichment analysis revealed that DEGs in each group were mainly associated with extracellular matrix, tissue development and skin development. KEGG analysis showed that the DEGs in each group during wound healing were mainly enriched in extracellular matrix-receptor interactions, focal adhesion and apoptosis pathways; while the DEGs after pressure treatment were mainly enriched in PI3K-Akt and MAPK signaling pathway except the pathways mentioned. qRT-PCR showed that the expression patterns of 6 DEGs were consistent with RNA-seq analysis, confirming the reliability of RNA-seq result. Conclusions RNA-Seq analysis identified differentially expressed genes in animal model of scars during wound healing and pressure therapy, which provided experimental evidence for clinical scar treatment.

Establishment of basic data of Bama minipigs for embryo-fetal development toxicity study / 中国比较医学杂志

Objective To obtain the basic data of Bama minipigs and provide basic reference for embryo-fetal developmental toxicity study. Methods Pregnant minipigs were sacrificed at different days during the gestation period respectively. The examinations included necropsy,count of corporea lutea,live and dead implantations,fetal body weight, and external,visceral, and skeletal examination of fetuses. Results The basic data of Bama minipigs, such as body weight, fetal development, and fetal malformation/variation were obtained. Conclusions We obtained the basic reproductive parameters of pregnant Bama minipigs and the indexes of fetal development, which can provide valuable reference data for embryo-fetal developmental toxicity tests of Bama minipigs.

Magnetic resonance angiography for head and neck arteries of bama minipig / 第二军医大学学报

Objective To display the normal vascular structures of head and neck of Bama minipig by three-dimensional time-of-flight (3D TOF) and time-resolved imaging of contrast kinetics (TRICKS) magnetic resonance angiography (MRA), and to compare and analyze the imaging indicators of arteries, so as to provide references for large animal models of cerebrovascular disease. Methods 3D TOF and TRICKS MRA of head and neck vessels in 3 healthy Bama minipigs were performed using a 3.0 T magnetic resonance system. The artery definition scores of the two MRA methods were compared, and the contrast ratio (CR), signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) of the main arteries were calculated for quantitative analysis. Results In terms of artery definition, 3D TOF MRA and TRICKS MRA both achieved high scores for the common carotid arteries and external carotid arterial system, but low scores for posterior circulation. 3D TOF MRA was significantly better than TRICKS MRA in displaying the intracranial artery system (1.60±0.50 vs 1.37±0.49, P＝0.019 8). Quantitative analysis showed that TRICKS MRA improved CR of arteries to different extents; however, there were no significant differences in SNR or CNR between 3D TOF and TRICKS MRA (P0.05). Conclusion Both 3D TOF and TRICKS MRA have good and comparable diagnostic performance for common carotid arteries and their main branch structures of Bama minipig, and each method has its advantages and limitations.

Alcoholization of pyogenichepatic abscess with absolute alcohol in Bama minipigs.

Pyogenic hepatic abscess (PHA) is a rare, but potentially serious disease. At present, ultrasound-guided or computed tomography-guided percutaneous needle aspiration or catheter drainage is appropriate as a first-line treatment. However, it is difficult to aspirate or drain pus and to select the appropriate antibiotic therapy if the abscess consists of thick pus and polymicrobial confections, or its pathogenic bacterium is multidrug resistant and cryptogenic. Case studies of alcoholization provide a novel method to manage PHA. However, the efficacy and safety of this treatment should be further evaluated. In the present study, the therapeutic efficacy and complications of alcoholization for PHAs in Bama minipigs were investigated. PHAs were prospectively treated by ultrasound-guided percutaneous instillation of absolute alcohol in the abscess cavity. The criteria for considering a successful intervention were met in all minipigs subsequent to alcoholization twice within 14 days. The procedures were well tolerated in all animals, and there were no alcoholic adverse effects or procedure-associated complications. In conclusion, ultrasound-guided percutaneous alcoholization is a safe and effective procedure to manage PHA. The problems of thick pus aspiration and selection of an appropriate antibiotic observed in other treatments were resolved effectively using alcoholization. This technique may reduce the treatment period and possibly become a novel strategy for the management of PHA.

[A Study on the Feasibility to Assay Minipig Insulin Through Human Insulin Radioimmunoassay Kit].

OBJECTIVES: To investigate the feasibility of using human-specific insulin radioimmunoassay (Ins-RIA) kit to measure the concentrations of serum insulin in minipigs. METHODS: The Serum samples (219) of Bama minipigs were collected. The concentrations of serum insulin in Bama minipigs were measured by Ins-RIA kit and porcine-specific insulin enzyme linked immunosorbent assay (Ins-ELISA) kit, respectively. The linear regression analysis was undertaken in a randomly selected 175 samples (80% of total samples) to establish predictive equations between the concentrations of serum insulin measured by Ins-ELISA and the concentrations of serum insulin measured by Ins-RIA. The equations were then cross-validated in the remaining 44 samples (20% of total samples) that had not been included in the regression analysis. RESULTS: Measured insulin concentration was lower with the Ins-ELISA than that with the Ins-RIA [(15.32±15.50) µIU/mL vs. (32.31±21.74) µIU/mL, respectively, P<0.000 1]; The final predictive equation for the Ins-ELISA (µIU/mL) was equal to -7.29+0.70 × Ins-RIA(µIU/mL) ( R2=0.94). The differences between the predicted values and the actual measured values were 17.18%. CONCLUSIONS: Insulin values in Bama minipigs obtained from the Ins-ELISA and Ins-RIA are not equivalent and differ significantly. However, the insulin concentration by Ins-ELISA can be well estimated by Ins-RIA.

Adiponectin induced AMP-activated protein kinase impairment mediates insulin resistance in Bama mini-pig fed high-fat and high-sucrose diet.

OBJECTIVE: Adipose tissue is no longer considered as an inert storage organ for lipid, but instead is thought to play an active role in regulating insulin effects via secretion adipokines. However, conflicting reports have emerged regarding the effects of adipokines. In this study, we investigated the role of adipokines in glucose metabolism and insulin sensitivity in obese Bama mini-pigs. METHODS: An obesity model was established in Bama mini-pigs, by feeding with high-fat and high-sucrose diet for 30 weeks. Plasma glucose and blood biochemistry levels were measured, and intravenous glucose tolerance test was performed. Adipokines, including adiponectin, interleukin-6 (IL-6), resistin and tumor necrosis factor alpha (TNF-α), and glucose-induced insulin secretion were also examined by radioimmunoassay. AMP-activated protein kinase (AMPK) phosphorylation in skeletal muscle, which is a useful insulin resistance marker, was examined by immunoblotting. Additionally, associations of AMPK phosphorylation with plasma adipokines and homeostasis model assessment of insulin resistance (HOMA-IR) index were assessed by Pearce's correlation analysis. RESULTS: Obese pigs showed hyperglycemia, high triglycerides, and insulin resistance. Adiponectin levels were significantly decreased (p<0.05) and IL-6 amounts dramatically increased (p<0.05) in obese pigs both in serum and adipose tissue, corroborating data from obese mice and humans. However, circulating resistin and TNF-α showed no difference, while the values of TNF-α in adipose tissue were significantly higher in obese pigs, also in agreement with data from obese humans but not rodent models. Moreover, strong associations of skeletal muscle AMPK phosphorylation with plasma adiponectin and HOMA-IR index were obtained. CONCLUSION: AMPK impairment induced by adiponectin decrease mediates insulin resistance in high-fat and high-sucrose diet induction. In addition, Bama mini-pig has the possibility of a conformable model for human metabolic diseases.

Comparison of organ coefficients and expression of hypoxia-related genes in the Bama and Juema minipigs / 中国比较医学杂志

Objective To compare the organ coefficients and expressions of hypoxia-related genes in Bama and Juema pigs.Method Real-time quantitative PCR was used to detect the changes of hypoxia gene expressions in the heart,liver,spleen,lung,and kidney of Juema and Bama miniature pigs.Results The organ coefficients of kidney and spleen of Juema pigs were significantly lower than Bama miniature pigs (P<0.05 for both).The heart and lung coefficients of Juema pigs were significantly higher than that of Bama miniature pigs (P<0.05 for both).The VEGF and HIF-1α expressions in the lung and kidney in Juema pigs were significantly higher than Bama pigs (P<0.05 or P<0.01).Only the EPO expression in in the lung of Juema pigs was significantly higher than that of the Bama miniature pigs (P<0.05).Conclusions These results indicate that the variation in organ coefficients may be resulted from evolutionary factors such as adaptiveness to environmental physical and energy conditions,pathogens,and energy metabolism demands,etc.in combination.Juema miniature pigs showing a significantly higher expression of hypoxia-related genes than that in Bama minipigs indicate that it has a strong plateau adaptability by higher gene expressions.

Co-dependence of genotype and dietary protein intake to affect expression on amino acid/peptide transporters in porcine skeletal muscle.

A total of 96 barrows (48 pure-bred Bama mini-pigs representing fatty genotype, and 48 Landrace pigs representing lean genotype) were randomly assigned to either a low- or adequate-protein treatment diet. The experimental period commenced at 5 weeks of age and extended to the finishing period. After euthanasia, blood and skeletal muscle samples were collected from pigs at the nursery, growing, and finishing phases. Our results indicate that the concentrations of free AAs in the plasma and muscle decreased as the age of the pigs increased. In addition, a strain × growth phase interaction (P < 0.05) was observed for the free AA pool in the plasma and muscle. The low-protein diet upregulated (P < 0.05) the mRNA levels for T1R1/T1R3 involved in glutamate binding, but downregulated (P < 0.05) the mRNA levels for PAT1, PAT2, and ASCT2, which transport neutral AAs into muscles. Bama mini-pigs had higher (P < 0.05) mRNA levels for LAT1, SNAT2, and EAAC1, but a lower (P < 0.05) mRNA level for PepT1, compared with Landrace pigs. Collectively, our findings indicate that adequate provision of dietary protein plays an important role in regulating profiles of free AA pools and expression of key AA/peptide transporters/transceptors in a genotype- and tissue-specific manner.