Minipig Research Articles

Development and laboratory evaluation of a competitive ELISA for serodiagnosis of Nipah and Hendra virus infection using recombinant Nipah glycoproteins and a monoclonal antibody.

Nipah virus (NiV) and Hendra virus (HeV), of the genus Henipavirus, family Paramyxoviridae, are classified as Risk Group 4 (RG4) pathogens that cause respiratory disease in pigs and acute/febrile encephalitis in humans with high mortality. A competitive enzyme-linked immunosorbent assay (cELISA) using a monoclonal antibody (mAb) and recombinant NiV glycoprotein (G) was developed and laboratory evaluated using sera from experimental pigs, mini pigs and nonhuman primates. The test depends on competition between specific antibodies in positive sera and a virus-specific mAb for binding to NiV-G. Based on 1,199 negative and 71 NiV positive serum test results, the cutoff value was determined as 35% inhibition. The diagnostic sensitivity and specificity of the NiV cELISA was 98.58 and 99.92%, respectively. When testing sera from animals experimentally infected with NiV Malaysia, the cELISA detected antibodies from 14 days post-infection (dpi) and remained positive until the end of the experiment (28 dpi). Comparisons using the Kappa coefficient showed strong agreement (100%) between the cELISA and a plaque reduction neutralization test (PRNT). Because our cELISA is simpler, faster, and gives comparable or better results than PRNT, it would be an adequate screening test for suspect NiV and HeV cases, and it would also be useful for epidemiological surveillance of Henipavirus infections in different animal species without changing reagents.

The use of human-derived feeder layers for the cultivation of transplantable human epidermal cell sheet to repair second degree burn wounds.

Human epidermal cell sheet (human-ECS) is a feasible treatment option for wound injury. Traditionally, researchers often use murine 3T3 fibroblast cells as feeder layer to support human epidermal cell sheet grafts, thus increase risk to deliver animal-borne infection. To overcome the potential risks involved with xenotransplantation, we develop human foreskin fibroblast cell as feeder layer culture system and investigate the effects of human-ECS on second-degree burn wound healing in mini-pig in order to develop more effective and safer therapies to enhance wound healing in human. Human epidermal keratinocytes and fibroblasts were isolated from foreskin tissue and were co-cultured to manufacture human-ECS. The cell morphology was monitored with phase-contrast microscopy, the stem cell markers were assessed by flow cytometry, and by colony-forming efficiency (CFE) assay. The structure of human-ECS was observed by hematoxylin and eosin staining. Expression of cytokines in human-ECS was confirmed by enzyme-linked immunosorbent assay. Second-degree burn wounds were created on the dorsal of miniature pig to evaluate the effect of oil gauze, oil gauze combined with commercial epidermal growth factor (EGF) cream, and oil gauze combined with human-ECS. Wound healing rate, histological examination, and Masson staining were measured to observe the wound repair efficacy. Real-time PCR and Western blot were utilized to detect the expression level of EGF and interleukin 6 (IL-6). Stratified human-ECS with 6-7 layers of epidermal cells was successfully cultivated with human-derived feeder cells, in which epidermal cell highly expressed CD49f and CFE was 3%±0.45%. Application of human-ECS induced a higher wound healing rate than commerical EGF cream and oil gauze control. The expression of EGF in human-ECS group was higher than those in the other groups; however, the expression of IL-6 was significantly decreased at day14 by human-ECS treatment group. Human-derived feeder cells are suitable for cultivation of human-ECS, avoiding pathogen transmission. Human-ECS could enhance second-degree burn wound healing, and its promoting effect involved secreting a variety of cytokines to regulate tissue reparative process.

Bilateral ultrasound‐guided lateral quadratus lumborum block in a minipig undergoing ovariectomy

AbstractA 2‐year‐old Göttingen miniature pig was presented for elective ovariectomy. After premedication with ketamine 15 mg/kg and azaperone 2 mg/kg intramuscularly, general anaesthesia was induced with propofol 1.5 mg/kg intravenously. With the animal in sternal recumbency, a 15 × 15 cm area on the dorsolateral aspect of the flank at the level of the second lumbar vertebra (L2) was clipped and aseptically prepared. Using a linear transducer, the epaxial musculature as well as the quadratus lumborum and abdominal wall muscles were identified. Then, 0.6 ml/kg of 0.25% ropivacaine was injected bilaterally at the level of L2 using a ventral‐to‐dorsal in‐plane approach under ultrasound guidance. Anaesthesia was stable and uneventful. Predefined thresholds for nociception were not exceeded at any time. After recovery, regular pain evaluation using the Universidade Estadual Paulista (UNESP)‐Botucatu pig composite acute pain scale did not indicate the need for rescue analgesia up to 4 h after the block was performed.

Urolitíase em suíno: Relato de caso

Urolithiasis is a pathological process that is characterized by the presence of stones or concretions in the urinary system, which can affect any and all species of animal. The present study consists of a case report involving a mini pig breed, diagnosed with non-obstructive urolithiasis and who underwent surgical intervention for the invasive treatment of the condition. The materials and methods include tests and statements related to the patient's treatment stages, ranging from laboratory tests to the postoperative period. The results showed that there was a possibility of the patient evolving into an obstructive urolithiasis, with a penectomy being the ideal surgical procedure for the treatment of the swine. Furthermore, the study supports the discussion of the use of surgical techniques in small to non-conventional animals, enriching it based on the medical-veterinary literature available on urolithiasis in swine, and filling gaps about the knowledge of this phenomenon, including the interventions, protocols adopted, drugs administered and other processes and procedures for rehabilitation. The conclusions reached identified the need to carry out future studies on urolithiasis in swine, given a general lack of publications in the national and international scientific literature on this phenomenon.

Validation of a surgical drill with a haptic interface in spine surgery

Real haptics is a technology that reproduces the sense of force and touch by transmitting contact information with real objects by converting human movements and the feel of the objects into data. In recent years, real haptics technology has been installed in several surgical devices. A custom-made surgical drill was used to drill into the posterior lamina to verify the time required for penetration detection and the distance the drill advanced after penetration. A surgeon operated with the drill and the same aspects were measured and verified. All experiments were performed on female miniature pigs at 9 months of age with a mean body weight of 23.6 kg (range 9–10 months and 22.5–25.8 kg, n = 12). There were statistically significant differences in the average reaction time and the distance travelled after penetration between a handheld drill and the drill with the penetration detection function (p < 0.001). The reaction time to detect penetration and the distance after penetration were both significantly improved when compared with those of the handheld surgical drill without the penetration detection function, with mean differences of 0.049 ± 0.019 s [95% CI 0.012, 0.086 s] and 2.511 ± 0.537 mm [95% CI 1.505, 3.516 mm]. In this study, we successfully conducted a performance evaluation test of a custom-made haptic interface surgical drill. A prototype high-speed drill with a haptic interface accurately detected the penetration of the porcine posterior lamina.

Multipotent Mesenchymal Stromal Cells from Porcine Bone Marrow, Implanted under the Kidney Capsule, form an Ectopic Focus Containing Bone, Hematopoietic Stromal Microenvironment, and Muscles.

Multipotent mesenchymal stromal cells (MSCs) are an object of intense investigation due to their therapeutic potential. MSCs have been well studied in vitro, while their fate after implantation in vivo has been poorly analyzed. We studied the properties of MSCs from the bone marrow (BM-MSC) before and after implantation under the renal capsule using a mini pig model. Autologous BM-MSCs were implanted under the kidney capsule. After 2.5 months, ectopic foci containing bones, foci of ectopic hematopoiesis, bone marrow stromal cells and muscle cells formed. Small pieces of the implant were cultivated as a whole. The cells that migrated out from these implants were cultured, cloned, analyzed and were proven to meet the most of criteria for MSCs, therefore, they are designated as MSCs from the implant-IM-MSCs. The IM-MSC population demonstrated high proliferative potential, similar to BM-MSCs. IM-MSC clones did not respond to adipogenic differentiation inductors: 33% of clones did not differentiate, and 67% differentiated toward an osteogenic lineage. The BM-MSCs revealed functional heterogeneity after implantation under the renal capsule. The BM-MSC population consists of mesenchymal precursor cells of various degrees of differentiation, including stem cells. These newly discovered properties of mini pig BM-MSCs reveal new possibilities in terms of their manipulation.

Establishment of acute liver failure model in Tibetan miniature pig and verified by dual plasma molecular adsorption system.

Acute liver failure (ALF) is a severe liver disease with high morbidity and mortality rates. Animal models are important for research on ALF. This study aimed to establish a reproducible, Tibetan miniature pig model of D-galactosamine-induced ALF and verify it using a dual plasma molecular adsorption system (DPMAS). Tibet miniature pigs were randomly divided into four groups (A, B, C, D) after catheterization. D-galactosamine (D-gal) at 0.45, 0.40, 0.35, and 0.35 g/kg body weight, respectively, was injected through the catheter. Group D was treated with DPMAS 48 h after D-gal administration. Vital signs and blood index values were recorded every 12 h after D-gal administration. H&E, TUNEL, Ki67, and Masson staining tests were performed. After D-gal administration, Tibetan miniature pigs developed different degrees of debilitation, loss of appetite, and jaundice. Survival times of groups A, B, C, and D were 39.7 ± 5.9, 53.0 ± 12.5,61.3 ± 8.1, and 61 ± 7 h, respectively. Blood levels of ALT, AST, TBIL, ammonia, PT, and inflammation factors significantly increased compared with baseline levels in the different groups (Ps < 0.05). Pathological results revealed a clear liver cell necrosis positive correlation with D-gal dose. However, DPMAS did not increase the survival time in ALF, ammonia, or liver cell necrosis. We successfully established a reproducible Tibetan miniature pig model of d-galactosamine-induced ALF, and we believe that a dosage of 0.35 g/kg is optimal.

Bionic artificial penile Tunica albuginea

Bionic artificial penile Tunica albuginea

Copeptin Reflect Left Ventricular Systolic Function at Early Stage of Acute Myocardial Infarction in a Pig Model.

Measurement of biomarkers early after acute myocardial infarction (AMI) might provide a cost-effective and widely available tool to assess infarct severity, myocardial dysfunction, and clinical outcomes. We aimed to induce AMI in miniature pigs, measure the levels of serum biomarkers and global LV function dynamically and explore the release kinetics and optimal sampling time points of copeptin and its correlation with global LV function. We induced AMI in the experimental group using a closed-chest model. Left ventricular (LV) function was detected by dual-source computed tomography (DSCT) and serum copeptin was determined by ELISA. The serum copeptin levels were increased at 1 hour, peaked at 3 hours, gradually decreased after 6 hours, and returned to baseline 3 days after AMI. At 3 to 6 hours, the copeptin cutoff of 16.97 to 17.44 pmol/l had 100% sensitivity and 100% specificity (P ⩽ .001) for AMI. Serum copeptin levels at 3 hours and 3 days were negatively correlated with the 3-hours LVEF (P ⩽ .001), respectively. Serum copeptin levels change in time, and measurements at 3 to 6 hours after AMI had the highest predictive value.

Collagen biomaterials promote the regenerative repair of abdominal wall defects in Bama miniature pigs.

Due to adhesion and rejection of recent traditional materials, it is still challenging to promote the regenerative repair of abdominal wall defects caused by different hernias or severe trauma. However, biomaterials with a high biocompatibility and low immunogenicity have exhibited great potential in the regeneration of abdominal muscle tissue. Previously, we have designed a biological collagen scaffold material combined with growth factor, which enables a fusion protein-collagen binding domain (CBD)-basic fibroblast growth factor (bFGF) to bind and release specifically. Though experiments in rodent animals have indicated the regeneration function of CBD-bFGF modified biological collagen scaffolds, its translational properties in large animals or humans are still in need of solid evidence. In this study, the abdominal wall defect model of Bama miniature pigs was established by artificial operations, and the defective abdominal wall was sealed with or without a polypropylene patch, and unmodified and CBD-bFGF modified biological collagen scaffolds. Results showed that a recurrent abdominal hernia was observed in the defect control group (without the use of mesh). Although the polypropylene patch can repair the abdominal wall defect, it also induced serious adhesion and inflammation. Meanwhile, both kinds of collagen biomaterials exhibited positive effects in repairing abdominal wall defects and reducing regional adhesion and inflammation. However, CBD-bFGF-modified collagen biomaterials failed to induce the regenerative repair reported in rat experiments. In addition, unmodified collagen biomaterials induced abdominal wall muscle regeneration rather than fibrotic repair. These results indicated that the unmodified collagen biomaterials are a better option among translational patches for the treatment of abdominal wall defects.

Assessment of Hepatic Lesions After non-Thermal Tumor Ablation by Irreversible Electroporation in a Pig Model.

Irreversible electroporation (IRE) is a non-thermal and minimal invasive modality to ablate pathologic lesions such as hepatic tumors. Histological analysis of the initial lesions after IRE can help predict ablation efficacy. We aimed to investigate the histological characteristics of early hepatic lesions after IRE application using animal models. IRE (1500 V/cm, a pulse length of 100 μs, 60 or 90 pulses) was applied to the liver of miniature pigs. H&E and TUNEL staining were performed and analyzed. Ablated zones of pig liver were discolored and separated from the normal zone after IRE. Histologic characteristics of ablation zones included preserved hepatic lobular architecture with a unique hexagonal-like structure. Apoptotic cells were detected, and sinusoidal dilatation and blood congestion were observed, but hepatic arteries and bile ducts were intact around the ablation zones. The early lesions obtained by delivering monophasic square wave pulses through needle electrodes reflected typical histological changes induced by IRE. Therefore, it was found that the histological assessment of the early hepatic lesion after IRE can be utilized to predict the IRE ablation effect.

Evaluation of porcine intestinal organoids as an in vitro model for mammalian orthoreovirus 3 infection.

Mammalian orthoreovirus type 3 (MRV3), which is responsible for gastroenteritis in many mammalian species including pigs, has been isolated from piglets with severe diarrhea. However, the use of pig-derived cells as an infection model for swine-MRV3 has rarely been studied. This study aims to establish porcine intestinal organoids (PIOs) and examine their susceptibility as an in vitro model for intestinal MRV3 infection. PIOs were isolated and established from the jejunum of a miniature pig. Established PIOs were characterized using polymerase chain reaction (PCR) and immunofluorescence assays (IFAs) to confirm the expression of small intestine-specific genes and proteins, such as Lgr5, LYZI, Mucin-2, ChgA, and Villin. The monolayered PIOs and three-dimensional (3D) PIOs, obtained through their distribution to expose the apical surface, were infected with MRV3 for 2 h, washed with Dulbecco's phosphate-buffered saline, and observed. Viral infection was confirmed using PCR and IFA. We performed quantitative real-time reverse transcription-PCR to assess changes in viral copy numbers and gene expressions linked to intestinal epithelial genes and antiviral activity. The established PIOs have molecular characteristics of intestinal organoids. Infected PIOs showed delayed proliferation with disruption of structures. In addition, infection with MRV3 altered the gene expression linked to intestinal epithelial cells and antiviral activity, and these effects were observed in both 2D and 3D models. Furthermore, viral copy numbers in the supernatant of both models increased in a time-dependent manner. We suggest that PIOs can be an in vitro model to study the infection mechanism of MRV3 in detail, facilitating pharmaceutical development.

Direct evidence of hydrogen absorption from the skin – a pig study

IntroductionIt has not been experimentally proven whether hydrogen gas (H2) is absorbed into the body through the skin by hydrogen-rich hot-water bathing.Material and methodsIn this study, Hairless mini pigs, whose skin closely resembles that of humans, were bathed in hydrogen (H2)-rich hot water to assess the absorption of H2 through the skin. An H2-rich water generation line was developed to maintain a high concentration of H2 via the circulation of hot water in an 80-litre bathtub. Two hairless mini pigs (14.2 ±1.4 kg, 60 days old, 1 male and 1 female) were first placed in the H2-dissolved bath. After a washout period, one pig was bathed in an H2-dissolved bath and the other in a bath containing no H2 for 20 min. During the experiment, blood was collected from the pigs’ jugular vein, carotid artery, inferior vena cava (IVCs), and portal vein to measure the blood H2 concentration.ResultsThe H2 concentration at the IVC of the pig in the H2-dissolved bath increased from 0.733 ±0.636 ppb (w/w) to 16.9 ±4.46 ppb (w/w) after 2 min, 37.2 ±13.8 ppb (w/w) after 10 min, and 45.7 ±7.73 ppb (w/w) (H2 saturation level: 3%) after 20 min. The blood H2 concentration levels of the pig in the non-H2 bath remained below the detection limit of 0.3 ppb.ConclusionsBathing in water with a high concentration of dissolved hydrogen was considered an effective means of supplying H2 to skin tissues and beyond.

Morphometric Changes of Osteocyte Lacunar in Diabetic Pig Mandibular Cancellous Bone.

Osteocytes play an important role in bone metabolism. The interactions of osteocytes with the surrounding microenvironment can alter cellular and lacunar morphological changes. However, objective quantification of osteocyte lacunae is challenging due to their deep location in the bone matrix. This project established a novel method for the analytical study of osteocytes/lacunae, which was then used to evaluate the osteocyte morphological changes in diabetic pig mandibular bone. Eight miniature pigs were sourced, and diabetes was randomly induced in four animals using streptozotocin (STZ) administration. The mandibular tissues were collected and processed. The jawbone density was evaluated with micro-CT. Osteocyte lacunae were effectively acquired and identified using backscattered electron scanning microscopy (BSE). A significantly decreased osteocyte lacunae size was found in the diabetic group. Using the acid etching method, it was demonstrated that the area of osteocyte and lacunae, and the pericellular areas were both significantly reduced in the diabetes group. In conclusion, a standard and relatively reliable method for analyzing osteocyte/lacunae morphological changes under compromised conditions has been successfully established. This method demonstrates that diabetes can significantly decrease osteocyte/lacunae size in a pig's mandibular cancellous bone.

Peptide Vaccine Against ADAMTS-7 Ameliorates Atherosclerosis and Postinjury Neointima Hyperplasia.

The metalloprotease ADAMTS-7 (a disintegrin and metalloproteinase with thrombospondin type 1 motif 7) is a novel locus associated with human coronary atherosclerosis. ADAMTS-7 deletion protects against atherosclerosis and vascular restenosis in rodents. We designed 3 potential vaccines consisting of distinct B cell epitopic peptides derived from ADAMTS-7 and conjugated with the carrier protein KLH (keyhole limpet hemocyanin) as well as aluminum hydroxide as an adjuvant. Arterial ligation or wire injury was used to induce neointima in mice, whereas ApoE-/- and LDLR-/- (LDLR [low-density lipoprotein receptor]) mice fed a high-fat diet were applied to assess atherosclerosis. In addition, coronary stent implantation was performed on vaccine-immunized Bama miniature pigs, followed by optical coherence tomography to evaluate coronary intimal hyperplasia. A vaccine, ATS7vac, was screened out from 3 candidates to effectively inhibit intimal thickening in murine carotid artery ligation models after vaccination. As well, immunization with ATS7vac alleviated neointima formation in murine wire injury models and mitigated atherosclerotic lesions in both hyperlipidemic ApoE-/- and LDLR-/- mice without lowering lipid levels. Preclinically, ATS7vac markedly impeded intimal hyperplasia in swine stented coronary arteries, but without significant immune-related organ injuries. Mechanistically, ATS7vac vaccination produced specific antibodies against ADAMTS-7, which markedly repressed ADAMTS-7-mediated COMP (cartilage oligomeric matrix protein) and TSP-1 (thrombospondin-1) degradation and subsequently inhibited vascular smooth muscle cell migration but promoted re-endothelialization. ATS7vac is a novel atherosclerosis vaccine that also alleviates in-stent restenosis. The application of ATS7vac would be a complementary therapeutic avenue to the current lipid-lowering strategy for atherosclerotic disease.

Endoscopic ultrasound-guided gallbladder endoscopic mucosal resection: a pilot porcine study

Introduction Endoscopic ultrasound (EUS)-guided natural orifice transluminal gallbladder polypectomy provides a minimally invasive alternative to cholecystectomy. The study aimed to investigate the feasibility and safety of protocol for gallbladder endoscopic mucosal resection (gEMR) under EUS guidance using a porcine model. Material and methods Fifteen Bama mini pigs were randomly divided into the control (CG, n = 3) and experimental (EG, n = 12) groups. EUS-guided fine needle aspiration was performed in the CG and used to establish a gallbladder pathway for polyp resection under EUS guidance in the EG. Procedural safety was evaluated using routine blood and biochemical tests, microbial bile cultures, histopathological tests, and enzyme-linked immunosorbent assays for inflammatory adhesion factors. Results EUS-guided metal stents were successfully deployed in all 12 pigs. Two cases of stent displacement occurred postoperatively, and one pig died of infectious peritonitis on the first day after stent implantation. In 11 surviving experimental animals, mature gallbladder paths were formed at 7–14 days after gastro-cholecystostomy, through which gEMR of gallbladder polyps was successfully performed. There were no significant changes in levels of inflammatory and adhesion factors during the postoperative process. Conclusions EUS-gEMR may be a safe and effective minimally invasive treatment approach for gallbladder polyps.

An electrochemical nitric oxide generator for in-home inhalation therapy in pulmonary artery hypertension

BackgroundInhaled NO is a selective pulmonary vasodilator proven to be therapeutic for patients with pulmonary artery hypertension (PAH). The most common NO delivery system in clinical practice is cylinder-based, but unfortunately limited by its high costs, complicated delivery, and the requirement of an extensive supply chain, leaving vast unmet medical needs globally.MethodsTo address the need for rapid, affordable, and safe production of nitric oxide (NO) for in-home inhalation therapy in patients with PAH. We developed a novel portable device to derive NO from a nitrite complex solution with a copper(II)-ligand catalyst, and further examined its effectiveness in a porcine model of PAH. This model was established by using female Bama miniature pig and induced by monocrotaline (MCT) administration.ResultsThis generator could rapidly and safely produce therapeutic NO at concentrations ranging from 0 to 100 parts per million (ppm) with the least disproportionated nitrogen dioxide (NO2) and byproducts. It could effectively alleviate pulmonary arterial pressure (PAP) and pulmonary vascular resistance (PVR) in piglets with PAH, without causing major physiologic disruptions.ConclusionsOur electrochemical NO generator is able to produce the desired NO doses for pulmonary vasodilation in a safe and sustainable way, with low costs, which paves the way for its subsequent clinical trials in the patient with PAH and other common cardiopulmonary conditions with a high disease burden around the world.

Analytical method development and dermal absorption of 2-amino-5-nitrophenol (2A5NP), a hair dye ingredient under oxidative condition.

Although 2-amino-5-nitrophenol (2A5NP) is one of the ingredients of hair dye, there has been no information on the dermal absorption rate of 2A5NP. 2A5NP is managed at less than 1.5% in Korea and Japan. In this study, analytical methods were developed and validated using high-performance liquid chromatography (HPLC) in various matrices of wash, swab, stratum corneum (SC), skin (dermis + epidermis), and receptor fluid (RF). Validation results were acceptable based on Korea Ministry of Food and Drug Safety (MFDS) guideline. The HPLC analysis showed a good linearity (r2 = 0.9992-0.9999), a high accuracy (93.1-110.2%), and a good precision (1.1-8.1%) in accordance with the validation guideline. Franz diffusion cell was used to determine dermal absorption of 2A5NP using mini pig skin. 2A5NP (1.5%) was applied to skin at 10μl/cm2. For certain cosmetic ingredients such as hair dye with short exposure time, an interimwash step (after 30min) was added during the study. After application for 30min and 24h, skin was wiped off with swab and SC was collected using tape stripping. RF was sampled at 0, 1, 2, 4, 8, 12, and 24h. Total dermal absorption rate of 2A5NP (1.5%) was determined to be 13.6 ± 2.9%.

Analysis of Variations in the Basic Indicators of Mini Pigs under Extreme Physical Exertion

Extreme physical exertion (work to failure) was simulated in untrained mini pigs. The results of complete blood tests, lactate measurements and clinical urine analysis revealed variations in the basic parameters of animals similar to those observed in people after extreme exertion.

THE STUDY ON THE EFFECT OF ARTIFICIAL OSTEOPOROSIS CREATED BY COMBINED OVARIECTOMY AND CALCIUM-RESTRICTED DIETS IN A PORCINE MODEL

This study design is to evaluate the mid-term changes in bone mineral density (BMD) with combined calcium-restricted and ovariectomized miniature porcine models as a large animal model in osteoporosis. The combined old practice hangs on for almost 30 years. Four 6-month-old (T0) female miniature pigs were enrolled in this study. The pigs were fed a normal diet prior to the ovariectomy at the age of 1 year and 3 months (T1) but switched to a diet with restricted calcium content afterwards. Each of the pigs received dual-energy X-ray absorptiometry (DXA) once before ovariectomy, and once every three months (T2, T3, T4) after the ovariectomy to evaluate the changes in BMD. The body weight of all four subject pigs increased significantly during this study ([Formula: see text]). The initial changes in both the BMD levels (T1/T2) were found to be statistically insignificant ([Formula: see text] and [Formula: see text], respectively). However, upon comparison of later BMD changes (T3/T4, T1/T3 and T1/T4), statistically significant elevations were found ([Formula: see text] for all three comparisons). Ovariectomy and calcium-restricted diets are ineffective in achieving an osteoporotic porcine model based on BMD assessments. BMD levels of the subject pigs continued to rise until the point at which body growth had stopped because the ideal pigs for surgical experiments were far from maturity. This finding is not unexpected; after all, the subject pigs are not senile. Without violations of the physiology and Institutional Animal Care and Use Committee (IACUC) regulations, moreover, pigs could be fed by strictly calcium-restricted diets or deprived of soybean component feed. Furthermore, the alternative protocols in osteoporotic porcine model shall perform experiments as soon as possible after ovariectomy. We should take other studies about artificial osteoporotic pigs more into consideration whether it is based on a rational method.