Effects of Succinate on Growth Performance, Meat Quality and Lipid Synthesis in Bama Miniature Pigs.

Succinate, one of the intermediates of the tricarboxylic acid cycle, is now recognized to play a role in a broad range of physiological and pathophysiological settings, but its role in adipogenesis is unclear. Our study used Bama miniature pigs as a model to explore the effects of succinate on performance, meat quality, and fat formation. The results showed that adding 1% succinate significantly increased the average daily gain, feed/gain ratio, eye muscle area, and body fat content (p < 0.05), but had no effect on feed intake. Further meat quality analysis showed that succinate increased the marbling score and intramuscular fat content of longissimus dorsi muscle (LM), while decreasing the shear force and the cross-sectional area of LM (p < 0.05). Metabolomics analysis of LM revealed that succinate reshaped levels of fatty acids, triglycerides, glycerophospholipids, and sphingolipids in LM. Succinate promotes adipogenic differentiation in porcine primary preadipocytes. Finally, dietary succinate supplementation increased succinylation modification rather than acetylation modification in the adipose tissue pool. This study elucidated the effects of succinate on the growth and meat quality of pigs and its mechanism of action and provided a reference for the role of succinate in the nutrition and metabolism of pigs.

Alternative Biological Material for Tissue Engineering of the Vagina: Porcine-Derived Acellular Vaginal Matrix.

BACKGROUND: Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is a severe congenital disorder characterized by vaginal hypoplasia caused by dysplasia of the Müllerian duct. Patients with MRKH syndrome often require nonsurgical or surgical treatment to achieve satisfactory vaginal length and sexual outcomes. The extracellular matrix has been successfully used for vaginal reconstruction. METHODS: In this study, we developed a new biological material derived from porcine vagina (acellular vaginal matrix, AVM) to reconstruct the vagina in Bama miniature pigs. The histological characteristics and efficacy of acellularization of AVM were evaluated, and AVM was subsequently transplanted into Bama miniature pigs to reconstruct the vaginas. RESULTS: Macroscopic analysis showed that the neovaginas functioned well in all Bama miniature pigs with AVM implants. Histological analysis and electrophysiological evidence indicated that morphological and functional recovery was restored in normal vaginal tissues. Scanning electron microscopy showed that the neovaginas had mucosal folds characteristics of normal vagina. No significant differences were observed in the expression of CK14, HSP47, and α-actin between the neovaginas and normal vaginal tissues. However, the expression of estrogen receptor (ER) was significantly lower in the neovaginas than in normal vaginal tissues. In addition, AVM promoted the expression of ß-catenin, c-Myc, and cyclin D1. These results suggest that AVM might promotes vaginal regeneration by activating the ß-catenin/c-Myc/cyclin D1 pathway. CONCLUSION: This study reveals that porcine-derived AVM has potential application for vaginal regeneration.

Gene cloning, prokaryotic expression and antiviral activities of interferon-αω from Chinese Bama miniature pigs.

Porcine interferon (PoIFN) complex represents an ideal model for studying IFN evolution that resulted from viral pressure during domestication. IFN-αω is an emergent subtype of type I IFNs which has been primarily characterized in domestic pigs. In this study, the PoIFN-αω cDNA was cloned from Chinese Bama miniature pigs by RT-PCR, and its tissue expression profile was analyzed by real-time RT-PCR. The cDNA was expressed in Escherichia coli as a His-tagged protein and purified by nickel affinity chromatography. The antiviral activities of recombinant PoIFN-αω (rPoIFN-αω) against four different pig viruses were measured using cytopathic effect (CPE) inhibition assay. Although the PoIFN-αω sequence of Bama miniature pigs was identical to that of domestic pigs, the tissue expression profiles differed significantly between the two pig species. The rPoIFN-αω showed dose-dependent pre-infection antiviral activities against porcine pseudorabies virus, vesicular stomatitis virus and porcine reproductive and respiratory syndrome virus, but not against porcine circovirus type 2. When used as treatment post infection with the three viruses, rPoIFN-αω showed the efficacy in decreasing CPE in the infected cells in a time-dependent manner. Therefore, the expressed rPoIFN-αω could be used as an antiviral agent against pig virus infections.

Serum metabolomics of Bama miniature pigs bitten by Bungarus multicinctus.

Bungarus multicinctus is one of the top ten venomous snakes in China, and its bite causes acute and severe diseases, but its pathophysiology remains poorly elucidated. Thus, an animal model of Bungarus multicinctus bite was established by intramuscular injection of 30µg/kg of Bungarus multicinctus venom, and then the serum metabolites were subsequently screened, identified and validated by ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF-MS) methods to explore the potential biomakers and possible metabolic pathways. Untargeted metabolomics analysis showed that 36 and 38 endogenous metabolites levels changed in ESI+ and ESI-, respectively, KEGG pathway analysis showed that 5 metabolic pathways, including mineral absorption, central carbon metabolism in cancer, protein digestion and absorption, aminoacyl-tRNA biosynthesis and ABC transporters might be closely related to Bungarus multicinctus bite. Targeted metabolomics analysis showed that there were significant differences in serum D-proline, L-leucine and L-glutamine after Bungarus multicinctus bite (P < 0.05). In addition, receiver operating characteristic (ROC) analysis showed that the diagnostic efficiency of L-Glutamine was superior to other potential biomarkers and the AUC value was 0.944. Moreover, we found evidence for differences in the pathophysiology of glutamine between Bungarus multicinctus bite group and normal group, specifically with the content of glutamine synthetase (GS) and glutaminase (GLS). Taken together, the current study has successfully established an animal model of Bungarus multicinctus bite, and further identified the links between the metabolic perturbations and the pathophysiology and the potential diagnostic biomakers of Bungarus multicinctus bite, which provided valuable insights for studying the mechanism of Bungarus multicinctus bite.

Porcine pancreas mesenchymal cell characterization and functional differentiation into insulin­producing cells in vitro.

Cell therapy is a promising treatment strategy for patients with type 1 diabetes. Porcine pancreas­derived mesenchymal stromal cells (PMSCs) have emerged as one of the most widely used cell resources owing to their high proliferative capacity and multi­lineage differentiation potential. Although the induction efficiency and insulin production of induced insulin­producing cells (IPCs) derived from PMSCs have been estimated, these have primarily focused on the function of induced cells and alterations in related gene expression levels. However, morphological analyses and biological characterization of PMSCs and induced IPCs have not been conducted. Therefore, the present study aimed to optimize an induction protocol, resulting in a 78.92% induction rate. The present study investigated the biological characteristics of PMSCs and optimized a simple but functional three­step protocol to transform PMSCs into IPCs. PMSCs were isolated from 2­3­month­old Bama miniature pig embryos, which were then subcultured to passage 16. The surface markers pancreatic and duodenal homeobox 1, NK6 homeobox 1, Vimentin, Nestin, CD73, CD90, neurogenin 3, CD45 and CD34 were detected by immunofluorescence staining or flow cytometry. Proliferative capacity was evaluated by constructing growth curves of cells at three different passages. Functional differentiation was assessed by morphological observation, dithizone staining, and immunofluorescence staining of C­peptide, insulin, NK6 homeobox 1 and glucagon. The production of insulin by differentiated cells was also analyzed by performing ELISAs. The results demonstrated that differentiated cells were distributed with an islet­like structure, expressed specific markers C­peptide and insulin, and displayed glucose responsiveness. The results of the present study demonstrated that PMSCs were functionally induced into IPCs with the optimized three­step protocol, which may serve as a potential cell therapy strategy to widen the availability and promote the clinical application of cell therapy.

Two dimensional automatic active shape model of degenerative disc repaired by low-intensity laser.

OBJECTIVE: Intervertebral disc degeneration is the main factor causing low back pain, and the related long-term treatment can improve the situation of degeneration. This study aimed to investigate the effect of low-intensity laser irradiation on the repair of degenerative intervertebral disc by two dimensional automatic active shape model (2D-AASM). METHODS: Nine Bama miniature pigs were randomly divided into three groups: control group (Con), model group (Mod) and laser treatment group (Las). After one month, the discs were treated with low-energy laser for another month. MRI was performed for one month, and the statistical shape model and 2D-AASM of intervertebral disc were established based on the minimum description length method. RESULTS: The model established by the proposed method is more accurate and the segmentation result is more accurate. From the segmented T2-weighted image, the signal intensity of the Mod group decreased significantly, and the signal intensity in the Las group was moderate and high compared with the Mod group. The HE staining display the structure of Con group was damaged, and the construction of Las group was restored compared with Mod group. CONCLUSIONS: The 2D-AASM method effectively improves the accuracy of intervertebral disc segmentation. The low-intensity laser has a protective effect on the repair of the degenerative intervertebral disc.

Photobiomodulation by diffusing optical fiber on spinal cord: A feasibility study in piglet model.

Previous studies on spinal cord injury (SCI) have confirmed that percutaneous photobiomodulation (PBM) therapy can ameliorate immunoinflammatory responses at sites of injury, accelerate nerve regeneration, suppress glial scar formation and promote the subsequent recovery of locomotor function. The current study was performed to evaluate a large-animal model employing implanted optical fibers to accurately irradiate targeted spinal segments. The method's feasibility and irradiation parameters that do not cause phototoxic reaction were determined, and the methodology of irradiating the spinal cord with near-infrared light was investigated in detail. A diffusing optical fiber was implanted above the T9 spinal cord of Bama miniature pigs and used to transfer near-infrared light (810 nm) onto the spinal cord surface. After daily irradiation with 200, 300, 500 or 1000 mW for 14 days, both sides of the irradiated area of the spinal cord were assessed for temperature changes. The condition of the spinal cord and the position of optical fiber were investigated by magnetic resonance imaging (MRI), and different parameters indicating temperature increases or phototoxicity were measured on the normal spinal cord surface due to light irradiation (ie, heat shock responses, inflammatory reactions and neuronal apoptosis), and the animals' lower-limb neurological function and gait were assessed during the irradiation process. The implanted device was stable inside the freely moving animals, and light energy could be directly projected onto the spinal cord surface. The screening of different irradiation parameters preliminary showed that direct irradiation onto the spinal cord surface at 200 and 300 mW did not significantly increase the temperature, stress responses, inflammatory reactions and neural apoptosis, whereas irradiation at 500 mW slightly increased these parameters, and irradiation at 1000 mW induced a significant temperature increase, heat shock, inflammation and apoptosis responses. HE staining of spinal cord tissue sections did not reveal any significant structural changes of the tissues compared to the control group, and the neurological function and gait of all irradiated animals were normal. In this study, we established an in-vivo optical fiber implantation method, which might be safe and stable and could be used to directly project light energy onto the spinal cord surface. This study might provide a new perspective for clinical applications of PBM in acute SCI.

Apolipoprotein E deficiency accelerates atherosclerosis development in miniature pigs.

Miniature pigs have advantages over rodents in modeling atherosclerosis because their cardiovascular system and physiology are similar to that of humans. Apolipoprotein E (ApoE) deficiency has long been implicated in cardiovascular disease in humans. To establish an improved large animal model of familial hypercholesterolemia and atherosclerosis, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 system (CRISPR/Cas9) was used to disrupt the ApoE gene in Bama miniature pigs. Biallelic-modified ApoE pigs with in-frame mutations (ApoEm/m ) and frameshift mutations (ApoE-/- ) were simultaneously produced. ApoE-/- pigs exhibited moderately increased plasma cholesterol levels when fed with a regular chow diet, but displayed severe hypercholesterolemia and spontaneously developed human-like atherosclerotic lesions in the aorta and coronary arteries after feeding on a high-fat and high-cholesterol (HFHC) diet for 6 months. Thus, these ApoE-/- pigs could be valuable large animal models for providing further insight into translational studies of atherosclerosis.

Feasibility study of acupuncture treatment on the miniature pig model of chronic myocardial ischemia / 中国比较医学杂志

Objective To explore the feasibility of needle-embedding therapy in the treatment of chronic myocardial ischemia using a miniature pig model established by placement of an Ameroid constrictor at the left anterior descending branch (LAD) of coronary artery in Bama miniature pigs during surgery. Methods The miniature pig model of chronic myocardial ischemia was established by placement of an Ameroid constrictor at the left anterior descending branch of the left coronary artery in Bama miniature pigs. The pig models were randomly divided into the treatment group (the"Neiguan " group) and the control group (the "Zusanli " group), and were treated with needle- embedding electroacupuncture at the"Neiguan" (PC6) and "Zusanli" (ST36) acupoints, respectively. Myocardial samples were taken at 6 weeks after surgery for light and electron microscopic examinations. Results Gross pathology showed that ischemic area in the myocardium appeared in both experimental groups. The ischemic area in the "Zusanli "group was larger than that of the"Neiguan"group. Histopathology showed that the acupuncture treatment at the"Neiguan"acupoint reduced the ischemic injury in the pig myocardial tissues. Ultrastructural observation of the myocardium showed mitochondrial vacuolization in cardiomyocytes and myocardial fibrosis in both groups. Conclusions Acupuncture therapy at the"Neiguan"acupoint of pericardial channel may exert protective effect on the myocardial ischemia by reducing the ischemia-injury of cardiomyocytes, but can not inhibit the already existed ischemia-induced cardiomyocytic injuries. Our findings suggest that the establishment of miniature pig model of chronic myocardial ischemia by surgically placing an Ameroid constrictor on the left anterior descending branch of left coronary artery and the needle-embedding in acupoints is feasible for the treatment of chronic myocardial ischemia in this pig model.

Establishment of a porcine model of acute liver failure after 85% hepatectomy / 中国实验动物学报

Objective To establish a porcine model of liver failure after different percent hepatectomy.Methods The porcine models of liver failure 75%,85%,95% hepatectomy were developed and the living conditions and survival time were recorded.The blood samples of pre-surgery,post-hepatectomy d1,d3,d5 and post-hepatectomy 1 week,2 weeks,and 3 weeks were collected for hepatic function analysis.Histological examination of liver tissues was performed using HE staining.Liver injury histology was interpreted and scored in the terminal samples.Results The average survival time of pigs with post-hepatectomy liver failure after 75%,85%,95% hepatectomy was 19.0±5.6 days,17.3±5.5 days,1.3±1.5 days,respectively.Their pathological scores were 5.67±0.52,8.17±0.82 and 8.50±0.71,respectively.With the increase of percent hepatic resection,the incidence of hepatic failure was increasing.ALT,AST,ALP,LDH and TBA were dramatically increased in the pigs after 85% hepatectomy.Conclusions The pig model of acute liver failure by 85% hepatectomy is successfully established,which can cause typical acute liver failure in Bama miniature pigs.

Effect of Soyabean Isoflavones Exposure on Onset of Puberty, Serum Hormone Concentration and Gene Expression in Hypothalamus, Pituitary Gland and Ovary of Female Bama Miniature Pigs.

This study was to investigate the effect of soyabean isoflavones (SIF) on onset of puberty, serum hormone concentration, and gene expression in hypothalamus, pituitary and ovary of female Bama miniature pigs. Fifty five, 35-days old pigs were randomly assigned into 5 treatment groups consisting of 11 pigs per treatment. Results showed that dietary supplementation of varying dosage (0, 250, 500, and 1,250 mg/kg) of SIF induced puberty delay of the pigs with the age of puberty of pigs fed basal diet supplemented with 1,250 mg/kg SIF was significantly higher (p<0.05) compared to control. Supplementation of SIF or estradiol valerate (EV) reduced (p<0.05) serum gonadotrophin releasing hormone and luteinizing hormone concentration, but increased follicle-stimulating hormone concentration in pigs at 4 months of age. The expression of KiSS-1 metastasis-suppressor (KISS1), steroidogenic acute regulatory protein (StAR) and 3-beta-hydroxysteroid dehydrogenase/delta-5-delta-4 isomerase (3ß-HSD) was reduced (p<0.01) in SIF-supplemented groups. Expression of gonadotropin-releasing hormone receptor in the pituitary of miniature pigs was reduced (p<0.05) compared to the control when exposed to 250, 1,250 mg/kg SIF and EV. Pigs on 250 mg/kg SIF and EV also showed reduced (p<0.05) expression of cytochrome P450 19A1 compared to the control. Our results indicated that dietary supplementation of SIF induced puberty delay, which may be due to down-regulation of key genes that play vital roles in the synthesis of steroid hormones.

Characterization of swine leukocyte antigen (SLA) polymorphism by sequence-based and PCR-SSP methods in Chinese Bama miniature pigs.

The highly polymorphic swine leukocyte antigen (SLA) genes have been repeatedly shown to influence swine immune traits, disease resistance, vaccine responsiveness and tumour penetrance. Analysis of the SLA diversity in as many pig breeds as possible is important to clarify the relationships between SLA genes and diseases or traits, and develop these pigs as valuable animal models for biomedical research. The Chinese Bama miniature pig breed is an economically significant breed that is available at several research institutions in China. In this study, we identified a total of 32 alleles at five polymorphic SLA loci (SLA-1, SLA-3, SLA-2, DRB1 and DQB1) representing nine class I and seven class II haplotypes using the reverse transcription polymerase chain reaction (RT-PCR) sequence-based typing (SBT) method. The possible functional sites of the SLA genes were predicted and analyzed by comparison with those of the human and mouse. Based on the sequence information, we subsequently developed a rapid PCR-based typing assay using sequence-specific primers (PCR-SSP) to efficiently follow the SLA types of the progeny. In the studied cohort (2n = 562), the most prevalent Haplotype Hp-35.6 (SLA-1(∗)1201, SLA-1(∗)1301-SLA-3(∗)0502-SLA-2(∗)1001-DRB1(∗)0501-DQB1(∗)0801) was identified in 182 Bama pigs with a frequency of 32.38%. The presence of the duplicated SLA-1 locus was confirmed in five of the class I haplotypes. Moreover, we identified two crossovers within the class I region and one between the class I and class II regions, which corresponded to recombination frequencies of 0.36% and 0.18%, respectively. The information of this study is essential for an understanding of the SLA allelic architecture and diversity, and it will be helpful for studying the adaptive immune response and further developing the more effective vaccines in the context of SLA specificities.