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

Background: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.Methods:The genomic DNAs of three Bama minipigs with T2DM,six sibling lowsusceptibility 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.Results: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.Conclusions: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.

Vestibular-evoked myogenic potentials recorded from miniature pigs and rats

Objective:To report vestibular evoked myogenic potentials from different recording sites(neck extensor or masseter muscles) in miniature pigs and rats.Methods:Potentials were recorded using 1000 Hz tone bursts from the neck extensor muscle or masseter muscle in normal adult Bama miniature pigs and rats anesthetized with 3%pentobarbital sodium and Sumianxin Ⅱ.Results:At 80 dB SPL,the first positive wave(P wave) of VEMPs was recognizable in 58%of rats with a latency of 6.45±0.23 ms and an amplitude of 1.45±0.49 μV when recorded from the neck extensor muscle,and in 50%of rats with a latency of 6.38±0.34 ms and an amplitude of 1.57±0.35 μV when recorded from the masseter muscle.In miniature pigs,at the same stimulus intensity,P wave was recognizable in 58%of the animals with a latency of 7.65±0.64 ms and an amplitude of 1.66±0.34 μV when recorded from the neck extensor muscle,and in 50%of the animals with a latency of 7.65±0.64 ms and an amplitude of 0.31±0.28 μV when recorded from the masseter muscle.Conclusion:VEMP can be induced from both neck extensor and masseter muscles in the miniature pig and rat.For a given species,the site of recording affects P wave induction rate and amplitude but not latency.Consistency and repeatability analysis suggests that the masseter muscle is a better recording site in miniature pigs while the cervical extensor is a better recording site in rats.For a given recording site,both latency and amplitude of the P wave are slightly greater in miniature pigs than in rats.

Cardiopulmonary,Biochemical and Haematological Effects of the Tiletamine/Zolazepam-Xylazine-Tramadol Combination to Provide Anaesthesia in Miniature Pigs

The objective of this study was to investigate the suitability of tiletamine/zolazepam-xylazine-tramadol combination for miniature pigs. Fourteen Chinese experimental miniature pigs subjected to this study received 3.5 mg tiletamine/zolazepam kg-1 bw, 1.32 mg xylazine kg-1 bw and 1.8 mg tramadol kg-1 bw intramuscularly, as a mixture of the drugs. Cardiopulmonary, biochemical and haematological parameters were recorded before drug administration and after anaesthesia. The combination of the compounds resulted in anaesthesia lasting about 87 min and a satisfactory immobilization for handling. Cardiopulmonary parameters were changed after administration, but there were within biologically acceptable limits. Biochemical and haematological values decreased after drug administration, however, they returned to the baseline at 24 h. At the doses described, tiletamine/zolazepam-xylazine-tramadol combination produced good immobilization in miniature pigs with minimal changes over time in cardiopulmonary, biochemical and haematological parameters.

Heat Stress Upregulates the Expression of TLR4 and Its Alternative Splicing Variant in Bama Miniature Pigs

Alternative splicing is a cellular mechanism in eukaryotes that results in considerable diversity ofgene products. It plays an important role in several diseases and cellular signal regulation. Heat stress is a major factor that induces immunosuppression in pigs. Little is known about the correlation between alternative splicing and heat stress in pigs. Therefore, this study aimed to clone, sequence and quantify the alternative splicing variant of toll-like receptor 4 （TLR4） in Bama miniature pigs （Sus scrofa domestica） following exposure to heat stress. The results showed that the second exon of TLR4 was spliced and 167 bp shorter in the alternative splicing variant, and the protein was putatively identified as a type of truncated membrane protein consisting of extramembrane, transmembrane and intramembrane regions lacking a signal peptide. Further, it was not a non- classical secretory protein. Five potential reference genes were screened for their potential as reliable standards to quantify the expression of TLR4 alternative spliced variants by real-time quantitative reverse-transcriptase polymerase chain reaction （qRT-PCR）. The stability of these reference genes was ranked using the geNorm and NormFinder programs, and ribosomal protein L4 （RPL4） and TATA box-binding protein （TBP） were found to be the two genes showing the most stable expression in the in vitro cultured peripheral blood mononuclear ceils （PBMCs） during heat shock. The mRNA level of the TLR4 gene （both classical and spliced） in stressed pigs increased significantly （P〈0.05）. Further, the expression levels of the alternative spliced variant of TLR4 （TLR4-ASV） showed a 2-3 folds increase in heat-stressed PBMCs as compared to control pigs. The results of the present study suggested that heat shock might modulate the host immune response by regulating the expressions of TLR4 and its alternative splicing variant.

Nucleus Transfer Efficiency of Ear Fibroblast Cells Isolated from Bama Miniature Pigs at Various Ages

Summary： Somatic cell nucleus transfer （SCNT） has been considered the most effective method for conserving endangered animals and expanding the quantity of adult animal models. Bama miniature pigs are genetically stable and share similar biological features to humans. These pigs have been used to establish animal models for human diseases, and for many other applications. However, there is a pan- city of studies on the effect of ear fibroblasts derived from different age of adult Bama miniature pigs on nucleus transfer （NT）. The present study examined the NT efficiency of ear fibroblasts from fetal, new- born, 1-, 2-, 4-, 6-, 12-month-old miniature pigs by using trypan blue staining, flow cytometry and NT technique, etc., and the cell biological function and SCNT efficiency were compared between groups. The results showed that ear fibroblasts grew well after passage in each group. Spindle-shaped cells ini- tially predominated, and gradually declined with increase of culture time and replaced by polygonal cells. Irregular cell growth occurred in the 2-month-old group and the elder groups. The growth curves of the ear fibroblasts were ＂S-shaped＂ in different age groups. The cell proliferation of postnatal ear fi- broblasts, especially those from 2-, 4-, 6-, 12-month-old miniature pigs was significantly different from that of fetus ear fibroblasts （P〈0.05 or P〈0.01）. Two-month- and 4-month-old ear fibroblasts had a sig- nificantly higher proportion of G1 stage cells （85% to 91%） than those at 6 and 12 months （66% to 74%, P〈0.01）. The blastocyst rate of reconstructed embryos originating from newborn, 1-, 2-, 4-month-old donor pigs was 6.06% to 7.69% with no significant difference from that in fetus fibroblast group （8.06%）. It was concluded that 〈4-month-old adult Bama miniature pigs represent a better donor cell resource than elder pigs.

Application of sodium alginate microspheres in ischemic stroke modeling in miniature pigs

The miniature pig is an optimal animal model for studying nervous system disease because of its physiologic and pathologic features. However, the rete mirabile composed of arteries and veins at the skull base limits their application as a model of ischemic stroke by middle cerebral artery occlusion. The present study investigated the possibility of establishing an ischemic stroke model in the miniature pig by blocking the skull base retia with sodium alginate microspheres. Three Bama miniature pigs were used. Using the monitor of C-arm X-ray machine, sodium aiginate microspheres （100-300 pm）, a novel embolic material, were injected through the femoral artery, aortic arch, common carotid artery, ascending pharyngeal artery and the retia. Results were evaluated using carotid arteriography, MRI, behavior observation and histology. The unilateral rete mirabile was completely blocked, resulting in disturbance in blood supply to the basal ganglia, astasia of the dght hind limb and salivation. MRI and hematoxylin-eosin staining showed an evident infarction focus in the basal ganglia. These findings indicate that sodium alginate microspheres are a suitable embolic material for blocking the skull base retia in miniature pigs to establish an ischemic stroke models.

Vestibular-evoked myogenic potentials in miniature pigs

Objective:To report detection of vestibular-evoked myogenic potentials (VEMPs) in the miniature pig. Methods:Potentials evoked by 1000 Hz tone bursts were recorded from neck extensor muscles and the masseter muscles in normal adult Bama miniature pigs anesthetized with 3%pentobarbital sodium and Carbachol II. Results:The latency of the first positive wave P from neck extensor muscles was 7.65 ± 0.64 ms, with an amplitude of 1.66 ± 0.34 uv and a rate of successful induction of 75%at 80 dB SPL. The latency of potentials evoked from the masseter muscles was 7.60 ± 0.78 ms, with an amplitude of 1.31 ± 0.28 uv and a rate successful induction of 66%at 80 dB SPL. Conclusion:The latencies and thresholds of VEMPs recorded from the neck extensor muscle and the masseter muscle appear to be comparable in normal adult Bama miniature pigs, although the amplitude recorded from the neck extensor muscle seems to be higher than that from the masseter muscle. However, because of their usually relatively superficial and easily accessible location, as well as their large volume and strong contractions, masseter muscles may be better target muscles for recording myogenic potentials.

Effects of simvastatin gel on bone regeneration in alveolar defects in miniature pigs

Currently, the most commonly used treatment methods for repairing alveolar furcation defects are periodontal guided tissue regeneration （GTR） and bone grafting. The objective of this study was to investigate the effects of simvastatin/methylcellulose gel on bone regeneration in alveolar defects in miniature pigs. Methods Alveolar defects were produced in 32 teeth （the third and fourth premolars） of 4 miniature pigs. The 32 experimental teeth were divided into 5 groups comprising control （C） and treatment （T） teeth： （1） empty defects without gel （group CO, n=4）; （2） defects injected with methylcellulose gel （group C1, n=4）; （3） defects injected with 0.5 rag/50 pl simvastatin/methylcellulose gel （group T1, n=8）; （4） defects injected with 1.5 mg/50 μl simvastatin/methylcellulose gel （group T2, n=8）; and （5） defects injected with 2.2 mg/50 μl simvastatin/methylcellulose gel （group T3, n=8）. Every week after surgery, the furcation sites were injected once with gel. At the eighth week afte（ surgery, the 4 pigs were sacrificed and underwent macroscopic observation, descriptive histologic examination, and regenerate bone quantitative histologic examination. Results At 8 weeks after surgery, the defect sites in the treatment groups were completely filled in with new bone and fibrous tissue. There was little new bone in the CO and C1 groups, and only a small number of osteoblasts and proliferative vessels could be seen on microscopic examination. Conclusions Miniature pigs are an ideal experimental animal for establishing a model of alveolar defects using a surgical method. Local application of simvastatin/methylcellulose gel can stimulate the regeneration of alveolar bone in furcation defect sites, because it promotes the proliferation of osteoblasts. The best dose of simvastatin gel to stimulate bone regeneration is 0.5 mg.

AN EXPERIMENTAL STUDY OF INTRAVASCULAR STENT IN ABDOMINAL AORTA OF ATHEROSCLEROTIC MINIATURE PIGS

To study the effects of nitinol alloy stent in the treatment of atherosclerotic arterial stenosis, eight miniature pigs were subject to abdominal aortic balloon denudation and high-fatty food. Then 8 nitinol alloy stents were implanted into each abdominal aortas of pigs. The pigs were equally divided into two groups. One group was given captopril (3 mg / kg / d). All animals were sacrified) for pathological examination 6 to 10 months after stent implantation. The degree of arterial intima proliferation in the areas of stent implantation not significantly different from that of areas of balloon denudation alone; the atherosclerotic lesions were found at the arterial surface of stent implantation sites. The intima layer was rich in smooth muscle cells, with atherosclerotic plaque formed around the stent wire. On the other hand, significant decrease in arterial intima proliferation was found in group Ⅱ with no atherosclerotic plaque. The arterial stenosis resulting from atherosclerotic lesion could not be prevented by implantation of intravascular stent, and on the contrary, the mechanical stress of stent wire might worsen the atherosclerosis. Captopril might impede the development of atherosclerotic stenosis after stent implantation.

Establishment of a model of Mycoplasma hyopneumoniae infection using Bama miniature pigs

Mycoplasma hyopneumoniae(M.hyopneumoniae),is the primary aetiological agent of enzootic pneumonia leading to chronic respiratory disease prevalent worldwide.Conventional pigs are the only animals used for pathogenicity studies and vaccine evaluations of M.hyopneumoniae.Considering that the challenge animals have better genetic stability and a smaller body size to operate with,an alternative experimental animal model of M.hyopneumoniae infection with Bama miniature pigs was established.Nine seven-week-old snatch-farrowed,porcine colostrum-deprived(SF-pCD)Bama miniature pigs and nine conventional pigs were randomly divided into two infected groups(Bama miniatureinfected(BI)and conventional-infected groups(CI),BI and CI,n=6)and two control groups(Bama miniature control(BC)and conventional control(CC)groups,BC and CC,n=3).Every piglet was tracheally inoculated with 5×10^(8) CCU/mL containing 10%suspension of a stock of frozen lung homogenate from SF-pCD pigs infected with virulent strain JS or sterilized KM2 medium.Typical lung lesions appeared in all infected pigs after necropsy,and the mean gross lung lesions was 17.3 and 13.7 in groups of BI and CI.Serum IgG and nasal sIgA antibody titres were increased significantly.Cilia shedding and mucus staining increased greatly in JS-infected bronchi.Obvious reddish gross lesions and M.hyopneumoniae antigen were detected,especially apparently observed in group of BI.Moreover,DNA copies of M.hyopneumoniae from bronchoalveolar lavage fluid(BALF)of each JS-infected piglet reached more than 10^(8),and M.hyopneumoniae could be re-isolated from each infected BALF.These results indicate that Bama miniature pigs could be used as an alternative and more maneuverable experimental infection model for M.hyopneumoniae and display typical clinical and pathological features consistent with those in conventional pigs.

Identification of the miniature pig inbred line by skin allograft

Skin grafting has been used as one of the most reliable tests to determine the genetic stability of laboratory animal such as mice and rats inbred line, but no identification of swine inbred lines by skin grafting has been reported. At present, Wuzhishan miniature pig （WZSP） inbred line has acquired the F24 individuals in China. In order to verify whether WZSP inbred line had D^en cultivated successfully, allogeneic skin grafts and related research were performed on F20 individuals of WZSP inbreeding population, compared with a control group of autologous transplantation. We observed the transplant recipients＇ wounds, detected peripheral blood-related indicators interleukin-2, 4 and 10, CD4~ and CD8~ lymphocytes, and conducted hematoxylin-eosin （HE） and Masson＇s staining of skin to judge whether the immune rejection reactions occurred within 28 days after transplantation. Chr. 7 genomic heterozygosity of 48 WZSP individuals from F20 to F22 was analyzed by high-density single nucleotide polymorphism （SNP） chips （60 000 SNPs）. The result showed that there were no significant differences in graft skin, the plasma interleukin-2, 4, 10, CD4~ and CD8~, HE and Masson＇s staining results between the allograft and autograft groups, and no immune rejection occurred on the allograft group. We found that 11 genes in Chr. 7 of major histocompatibility complex （MHC） I and MHC II were homozygous which confirmed that immune antibody of the allograft and autograft groups were highly identical and also provided a theoretical basis to no immune rejection occurred on the allograft in the inbred WZSP. The result proved that the WZSP inbred line had been cultivated successfully for the first time in the world. The test methods also provide a scientific basis for the identification of swine and mammal inbred lines.

Current Situation and Progress of Pig Model in Otology Research

Pig models are widely used in otological research. The establishment of common pig and miniature pig animal models has opened up new fields in otological research and is also an ideal large otological animal model. This article introduces the applications, current status, progress, advantages, and issues of using pigs as otologic animal models. It summarizes current research on pigs in the fields of hearing disorders, otitis, vertigo, cochlea, gene editing, and tissue engineering, among other otologic and audiological areas. These models are valuable for translating basic medical science into clinical applications. Based on this, platforms have been established for studying deafness and vertigo, cochlear implantation experiments, stem cell and gene therapy, and tissue engineering. These serve as ideal experimental models for the prevention and treatment of ear diseases, pointing toward new directions. This will bolster the promotion and application of pig models in the fields of tissue engineering and gene editing in the future.

MICRODISSECTION OF MINIATURE PIG EAR

Objective To investigate the suitability of miniature pigs as an animal model for otological research. Methods Microdissection of the temporal bone was performed on 10 miniature pigs and recorded on photo-graphs. Results The morphology and measurement of the external, middle and inner ear and the lateral re-cess of the miniature pigs were obtained by microdissection. Conclusion Compared to traditional animal models, the miniature pig may be a better model for biomedical research because of its many similarities in physiological functions with humans. Similarities of the temporal bone structures, including the external, middle and inner ear and the lateral recess, between the miniature pig and human make the animal a poten-tially useful model for otological research.

Application of Gas Chromatography-mass Spectrometry in Analyzing Pharmacokinetics and Distribution of Deltamethrin in Miniature Pig Tissues

Objective To characterize the pharmacokinetics and distribution profiles of deltamethrin in miniature pig tissues by gas chromatography-mass spectrometry （GC-MS）. Methods Pharmacokinetics and distribution of deltamethrin in blood and tissues of 30 miniature pigs were studied by GC-MS after oral administration of deltamethrin （5 mg/kg bw）. Data were processed by 3P97 software. Results The serum deltamethrin level was significantly lower in tissues than in blood of miniature pigs.The AUC0-72 h, Cmax, of deltamethrin were 555.330±316.987 ng h/mL and 17.861±11.129 ng/mL, respectively. The Tmax, of deltamethrin was 6.004±3.131 h.Conclusion The metabolism of deltamethrin in miniature pigs is fit for a one-compartment model with a weighting function of I/C^2. Deltamethrin is rapidly hydrolyzed and accumulated in miniature pig tissues.

Effect of Same-dose Single or Dual Field Irradiation on Damage to Miniature Pig Parotid Glands

Aim To evaluate the effect of single or dual field irra- diation （IR） with the same dose on damage to miniature pig parotid glands. Methodology Sixteen miniature pigs were divided into two IR groups （n=6） and a control group （n=4）. The irradi- ation groups were subjected to 20 Gy X-radiation to one parotid gland using single-field or dual-field modality by linear accelerator. The dose-volume distributions between two IR groups were compared. Saliva from parotid glands and blood were collected at 0, 4, 8 and 16 weeks after irradiation. Parotid glands were removed at 16 weeks to evaluate tissue morphology.Results The irradiation dose volume distributions were significantly different between single and dual field irradi- ation groups （t----4.177, P=0.002）, although dose volume histogramin （DVH） indicated the equal maximal dose in parotid glands. Saliva flow rates from IR side decreased dramatically at all time points in IR groups, especially in dual field irradiation group. The radiation caused changes of white blood cell count in blood, lactate dehydrogenase and amylase in serum, calcium, potassium and amylase in saliva. Morphologically, more severe radiation damage was found in irradiated parotid glands from dual field irradi- ation group than that from single field irradiation group. Conclusion Data from this large animal model demons- trated that the radiation damage from the dual field irradiation was more severe than that of the single field irradiation at the same dose, suggesting that dose-volume distribution is an important factor in evaluation of the radiobiology of parotid glands.

Transcriptomic and proteomic studies of condylar ossification of the temporomandibular joint in porcine embryos

Background:The ossification mechanism of the temporomandibular joint(TMJ)condyle remains unclear in human embryo.The size and structure of TMJ,shape of articular disc and the characteristics of omnivorous chewing in the pig are similar to those of humans.The pig is an ideal animal for studying the mechanism of ossification of the TMJ condyle during the embryonic period.Method:In a previous study by our group,it was found that there was no condylar ossification on embryonic day(E)45,but the ossification of condyle occurred between E75 and E90.In this study,a total of 12 miniature pig embryos on E45 and E85 were used.Six embryos were used for tissue sections(3 in each group).The remaining six embryos were used for transcriptomic and proteomic studies to find differential genes and proteins.The differentially expressed genes in transcriptome and proteomic analysis were verified by QPCR.Results:In total,1592 differential genes comprising 1086 up-regulated genes and 506 down-regulated genes were screened for fold changes of≥2 to≤0.5 between E45 and E85.In the total of 4613 proteins detected by proteomic analysis,there were 419 differential proteins including 313 up-regulated proteins and 106 down-regulated proteins screened for fold changes of≥2 to≤0.5 between E45 and E85.A total of 36 differential genes differing in both transcriptome and proteome analysis were found.QPCR analysis showed that 14 of 15 selected genes were consistent with transcriptome analysis.Conclusion:Condylar transcriptome and proteomic analysis during the development of TMJ in miniature pigs revealed the regulatory genes/proteins of condylar ossification.

Involvement of NLRP3-inflammasome pathway in noise-induced hearing loss

The inflammasome is a multiprotein oligomer in the cell cytoplasm and is part of the innate immune system.It plays a crucial role in the pathological process of noise-induced hearing loss(NIHL).However,the mechanisms of NLR family pyrin domain containing 3(NLRP3)inflammasome activation in NIHL have not been clearly demonstrated.In this study,miniature pigs were exposed to white noise at 120 dB(A)and auditory brainstem response measurements were used to measure their hearing function.Immunofluorescence staining,confocal laser scanning microscopy,western blot assay,and quantitative reverse transcription-polymerase chain reaction were used to analyze inflammasome-related protein distribution and expression.NLRP3,interleukin-1β,interleukin-18,and cleaved-caspase-1 were highly expressed in the cochlea after 120 dB(A)white noise exposure.Our findings suggest that NLRP3-inflammasomes in the cochlea may be activated after acoustic trauma,which may be an important mechanism of noise-induced hearing loss.

Morphological characteristics of submandibular glands of miniature pig

Background Miniature pig （minipig） is increasingly used as a large animal model for a variety of biomedical studies. Little information is available in the literature on anatomy, histology and sialogragh of the submandibular gland of the minipig. The purpose of this study was to characterize the morphology of a miniature pig＇s （minipig） submandibular gland as a large animal model for further biomedical studies. Methods Five minipigs were subjected to sialographic, anatomic, histologic, histochemical and uhrastructural evaluations for submandibular glands. Results Sialograms showed a long, horizontal main excretory duct and a pear-shaped gland located inferoposterior to the angle of the mandible. The submandibular glands lied superficial to the suprahyoid, and infrahyoid muscle groups, and were covered by the inferior portion of the parotid gland. The submandibular glands were characterized by a mixed parenchyma of mucous and serous secretory aeini. Alcian blue （AB） staining and periodic acid-Schiff （PAS） reactions demon-strated that minipig submandibular glands synthesized and secreted acid mucous substances by serous cells and polysaccharide, and neutral mucous substances, by mucous cells. Conclusion The submandibular gland of the minipig is considered a useful large salivary gland animal model for biomedical studies.

An Improved Urethral Catheterization in Female Pigs： A Pilot Study

With the popularity of urine flow dynamic monitoring and indwelling catheter technologies in animal experiments,the urethral catheterization has become a common technique in scientific research.The miniature pig is considered as one of the major animal species used in scientific research and is increasingly being used as an alternative to a dog or monkey as the nonrodent species of choice in the preclinical toxicological testing of pharmaceuticals.[1] Since there are three acute angles bending in the urethra of the male miniature pig,and the end of the penis head is cork-screw shaped,it is difficult to implement routine urethral catheterization in male miniature pigs.Currently,researchers usually implement bladder colostomies or dissections in male miniature pigs,while transurethral urethral catheterization can be implemented in female miniature pigs.According to the existing reports,implementing transurethral urethral catheterization in female miniature pigs is seldom seen.[2] We chose 3-month-old female Bama miniature pigs in our experiments and employ the following methods：first,use a ＂V＂-shaped platform with an angle of about 30° to the horizontal plane;second,utilize a pediatric laryngoscope for deep lighting;third,bend the lower limbs and press down to the ventral side.Satisfactory results through transurethral urethral catheterization were achieved in the end.