Transcriptomic analysis of testis and epididymis tissues from Banna mini-pig inbred line boars with single-molecule long-read sequencing†.

In mammals, testis and epididymis are critical components of the male reproductive system for androgen production, spermatogenesis, sperm transportation, as well as sperm maturation. Here, we report single-molecule real-time sequencing data from the testis and epididymis of the Banna mini-pig inbred line (BMI), a promising laboratory animal for medical research. We obtained high-quality full-length transcriptomes and identified 9879 isoforms and 8761 isoforms in the BMI testis and epididymis, respectively. Most of the isoforms we identified have novel exon structures that will greatly improve the annotation of testis- and epididymis-expressed genes in pigs. We also found that 3055 genes (over 50%) were shared between BMI testis and epididymis, indicating widespread expression profiles of genes related to reproduction. We characterized extensive alternative splicing events in BMI testis and epididymis and showed that 96 testis-expressed genes and 79 epididymis-expressed genes have more than six isoforms, revealing the complexity of alternative splicing. We accurately defined the transcribed isoforms in BMI testis and epididymis by combining Pacific Biotechnology Isoform-sequencing (PacBio Iso-Seq) and Illumina RNA Sequencing (RNA-seq) techniques. The refined annotation of some key genes governing male reproduction will facilitate further understanding of the molecular mechanisms underlying BMI male sterility. In addition, the high-confident identification of 548 and 669 long noncoding RNAs (lncRNAs) in these two tissues has established a candidate gene set for future functional investigations. Overall, our study provides new insights into the role of the testis and epididymis during BMI reproduction, paving the path for further studies on BMI male infertility.

Differentiation Capacity of Porcine Skeletal Muscle-Derived Stem Cells as Intermediate Species between Mice and Humans.

Large animal experiments are important for preclinical studies of regenerative stem cell transplantation therapy. Therefore, we investigated the differentiation capacity of pig skeletal muscle-derived stem cells (Sk-MSCs) as an intermediate model between mice and humans for nerve muscle regenerative therapy. Enzymatically extracted cells were obtained from green-fluorescence transgenic micro-mini pigs (GFP-Tg MMP) and sorted as CD34+/45- (Sk-34) and CD34-/45-/29+ (Sk-DN) fractions. The ability to differentiate into skeletal muscle, peripheral nerve, and vascular cell lineages was examined via in vitro cell culture and in vivo cell transplantation into the damaged tibialis anterior muscle and sciatic nerves of nude mice and rats. Protein and mRNA levels were analyzed using RT-PCR, immunohistochemistry, and immunoelectron microscopy. The myogenic potential, which was tested by Pax7 and MyoD expression and the formation of muscle fibers, was higher in Sk-DN cells than in Sk-34 cells but remained weak in the latter. In contrast, the capacity to differentiate into peripheral nerve and vascular cell lineages was significantly stronger in Sk-34 cells. In particular, Sk-DN cells did not engraft to the damaged nerve, whereas Sk-34 cells showed active engraftment and differentiation into perineurial/endoneurial cells, endothelial cells, and vascular smooth muscle cells, similar to the human case, as previously reported. Therefore, we concluded that Sk-34 and Sk-DN cells in pigs are closer to those in humans than to those in mice.

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.

Novel dapagliflozin di-L-proline cocrystal-loaded tablet: preparation, physicochemical characterization, and pharmacokinetics in beagle dogs and mini-pigs.

Dapagliflozin base and a commercial dapagliflozin propanediol hydrate cocrystal (DPF-PDHC) were highly hygroscopic and thermally unstable. In this study, to address this limitation, we prepared a novel dapagliflozin di-L-proline cocrystal (DPF-LPC) and evaluated its physicochemical characterization compared with DPF-PDHC. After the preparation of the DPF-LPC-loaded tablet, its dissolution, stability and bioequivalence in beagle dogs and mini-pigs were assessed. DPF-LPC was well prepared with a dapagliflozin base and L-proline in a molar ratio of 1:2. Similar to DPF-PDHC, DPF-LPC was highly lipophilic and crystalline in nature. However, these two cocrystals exhibited different melting points and crystalline structures, indicating their different cocrystal forms. Moreover, DPF-LPC exhibited less hygroscopicity and lower water content than DPF-PDHC. The DPF-LPC-loaded tablet composed of DPF-LPC, Comprecel M102, lactose monohydrate, crospovidone, magnesium stearate, and Opadry (coating) at a weight ratio of 15.6:104.4:100.0:8.0:2.0:7.0, was dissolution-equivalent to the commercial tablet. Moreover, it provided lower impurities than the commercial tablet, indicating its better stability. In the two animals, there were no significant differences in the plasma concentrations, AUC, Cmax, and Tmax values, suggesting that they were bioequivalent. Therefore, the novel DPF-LPC-loaded tablet with excellent stability and bioequivalence may be used as a potential alternative to the commercial DPF-PDHC-loaded tablet.

Consequences of parenteral iron-dextran loading investigated in minipigs. A new model of transfusional iron overload.

Patients with blood transfusion-dependent anemias develop transfusional iron overload (TIO), which may cause cardiosiderosis. In patients with an ineffective erythropoiesis, such as thalassemia major, common transfusion regimes aim at suppression of erythropoiesis and of enteral iron loading. Recent data suggest that maintaining residual, ineffective erythropoiesis may protect from cardiosiderosis. We investigated the common consequences of TIO, including cardiosiderosis, in a minipig model of iron overload with normal erythropoiesis. TIO was mimicked by long-term, weekly iron-dextran injections. Iron-dextran loading for around one year induced very high liver iron concentrations, but extrahepatic iron loading, and iron-induced toxicities were mild and did not include fibrosis. Iron deposits were primarily in reticuloendothelial cells, and parenchymal cardiac iron loading was mild. Compared to non-thalassemic patients with TIO, comparable cardiosiderosis in minipigs required about 4-fold greater body iron loads. It is suggested that this resistance against extrahepatic iron loading and toxicity in minipigs may at least in part be explained by a protective effect of the normal erythropoiesis, and additionally by a larger total iron storage capacity of RES than in patients with TIO. Parenteral iron-dextran loading of minipigs is a promising and feasible large-animal model of iron overload, that may mimic TIO in non-thalassemic patients.

Lentilactobacillus kribbianus sp. nov., isolated from the small intestine of a mini pig.

A Gram-stain-positive, facultative anaerobic, rod-shaped bacteria isolated from the small intestine of a mini pig was designated as strain YH-lac9T. 16S rRNA gene sequence analysis revealed that the strain belongs to the genus Lentilactobacillus and is closely related to Lentilactobacillus senioris JCM 17472T, Lentilactobacillus rapi JCM 15042T and Lentilactobacillus diolivorans JCM 13927T, with 97.6, 96.2 and 95.7 % sequence similarity, respectively. Analysis of housekeeping gene sequences (pheS and recA) revealed that the strain formed a sub-cluster with L. senioris, supporting the results of 16S rRNA gene sequences analysis. The average nucleotide identity value for YH-lac9T and the most closely related strain is 74.1 %. The main fatty acids are C18 : 1ω9c, summed feature 7, C16 : 0 and summed feature 8. The G+C content of the genomic DNA is 37.8 mol%. In view of its chemotaxonomic, phenotypic and phylogenetic properties, YH-lac9T (=KCTC 25005=JCM 33997) represents a novel taxon. The name Lentilactobacillus kribbianus sp. nov. is proposed.

Clostridium vitabionis sp. nov., isolated from the large intestine of a mini-pig.

An obligately anaerobic, Gram-stain-negative, spore-forming, short rod-shaped bacterium, designated strain YH- T4B42T, was isolated from the large intestine of a mini-pig. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belongs to the genus Clostridium and is most closely related to Clostridium aminophilum KCTC 5424T, Clostridium symbiosum KCTC 15329T and Clostridium butyricum KCTC 1871T, with 95.5, 92.4 and 83.0 % sequence similarity, respectively. The average nucleotide identity values for strain YH-T4B42T and the closest related strains were lower than 72 %. The G+C content of the isolate was 55.8 mol%. The cell-wall peptidoglycan was A1γ type and contained meso-diaminopimelic acid. The predominant fatty acids were C16 : 0, C18 : 1 cis 9, C14 : 0 and C18 : 0. The major end products of glucose fermentation were lactate, formate and acetate, with a minor amount of butyrate. Based on its phenotypic, phylogenetic and chemotaxonomic properties, a novel species, Clostridium vitabionis sp. nov., is proposed for strain YH-T4B42T (=KCTC 25105T=NBRC 114767T).

Lacticaseibacillus absianus sp. nov., isolated from the cecum of a mini-pig.

A rod-shaped, facultative anaerobic, Gram-stain-positive bacteria, isolated from the cecum of a mini-pig, was designated as strain YH-lac23T. Analysis of 16S rRNA gene sequences revealed that the strain was closely related to Lacticaseibacillus daqingensis JCM 33273T (97.9 %), Lacticaseibacillus porcinae KCTC 21027T (96.2 %) and Lacticaseibacillus manihotivorans KCTC 21010T (95.7 %). Analysis of housekeeping gene sequences (pheS and recA) revealed that the strain formed a sub-cluster with L. daqingensis. The average nucleotide identity value for YH-lac23T and its most closely related strain (L. daqingensis) is 80.7 %. The main fatty acids are C18 : 1ω9c and C16 : 0. The cell wall contains the peptidoglycan of meso-diaminopimelic acid. The G+C content of the genomic DNA is 59.8 mol%. In view of the chemotaxonomic, phenotypic and phylogenetic properties, YH-lac23T (=KCTC 25006=JCM 33998) represents a novel taxon. The name Lacticaseibacillus absianus sp. nov. is proposed.

The effects of growth factors on proliferation of spermatogonial stem cells from Guangxi Bama mini-pig.

The objective of this study was to investigate the effects of different growth factors on the proliferation of Bama mini-pig spermatogonial stem cells (SSCs) in vitro. The growth factors glial cell line-derived neurotrophic factor (GDNF), leukaemia inhibitory factor (LIF), GDNF family receptor alpha-1 (GFRα1) and basic fibroblast growth factor (bFGF) were investigated. The SSCs were seeded on SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) feeder layers. Cultivation of the cells were subjected to a factorial design of the growth factors GDNF + bFGF, GDNF + bFGF + GFRα1, LIF + bFGF and LIF + bFGF + GFRα1. The SSCs could propagate for 25 passages in the medium adding GDNF + bFGF + GFRα1, 22 passages in the medium adding GDNF + bFGF, 6 passages in the medium adding LIF + bFGF, or LIF + bFGF + GFRα1. qRT-PCR analysis showed that the highest mRNA expression levels of NANOG, POU5F, DDX4, GFRα1 and UCHL1 were detected in the group adding GDNF + bFGF + GFRα1. The SSCs from the group adding GDNF + bFGF + GFRα1 also showed UCHL1-, DBA- and CDH1-positive staining. Moreover, Stra8 and Scp3 expression, and haploid peak were detected after induction of the SSCs from the group adding GDNF + bFGF + GFRα1. In conclusion, pig SSCs could be maintained for long term in the presence of GDNF, bFGF, and GFRα1.

The changes in glucose metabolism and cell proliferation in the kidneys of polycystic kidney disease mini-pig models.

The pathogenic mechanism of polycystic kidney disease (PKD) is unclear. Abnormal glucose metabolism is maybe involved in hyper-proliferation of renal cyst epithelial cells. Mini-pigs are more similar to humans than rodents and therefore, are an ideal large animal model. In this study, for the first time, we systematically investigated the changes in glucose metabolism and cell proliferation signaling pathways in the kidney tissues of chronic progressive PKD mini-pig models created by knock-outing PKD1gene. The results showed that in the kidneys of PKD mini-pigs, the glycolysis is increased and the expressions of key oxidative phosphorylation enzymes Complexes I and IV significantly decreased. The activities of mitochondrial respiration chain Complexes I and IV significantly decreased; the phosphorylation level of key metabolism-modulating molecule AMP-activated protein kinase (AMPK) significantly decreased; and the mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase (ERK) signaling pathway are activated obviously. This study showed that in the kidneys of PKD mini-pigs, the level of glycolysis significantly increased, oxidative phosphorylation significantly decreased, and cell proliferation signaling pathways significantly activated, suggesting that metabolic changes in PKD may result in the occurrence and development of PKD through the activation of proliferation signaling pathways.

Multiparametric CMR imaging of infarct remodeling in a percutaneous reperfused Yucatan mini-pig model.

To further understanding of the temporal evolution and pathophysiology of adverse ventricular remodeling over the first 60 days following a myocardial infarction (MI) in both the infarcted and remote myocardium, we performed multi-parametric cardiac magnetic resonance (CMR) imaging in a closed-chest chronic Yucatan mini-pig model of reperfused MI. Ten animals underwent 90 min left anterior descending artery occlusion and reperfusion. Three animals served as controls. Multiparametric CMR (1.5T) was performed at baseline, Day 2, Day 30 and in four animals on Day 60 after MI. Left ventricular (LV) volumes and infarct size were measured. T1 and T2 mapping sequences were performed to measure values in the infarct and remote regions. Remote region collagen fractions were compared between infarcted animals and controls. Procedure success was 80%. The model created large infarcts (28 ± 5% of LV mass on Day 2), which led to significant adverse myocardial remodeling that stabilized beyond 30 days. Native T1 values did not reliably differentiate remote and infarct regions acutely. There was no evidence of remote fibrosis as indicated by partition coefficient and collagen fraction analyses. The infarct T2 values remained elevated up to 60 days after MI. Multiparametric CMR in this model showed significant adverse ventricular remodeling 30 days after MI similar to that seen in humans. In addition, this study demonstrated that remote fibrosis is absent and that infarct T2 signal remains chronically elevated in this model. These findings need to be considered when designing preclinical trials using CMR endpoints.

Sinus augmentation using a mini-pig model: Effect of ceramic and allogeneic bone biomaterials.

BACKGROUND: Present clinical practice broadly relies on off-the-shelf allogeneic, xenogeneic or synthetic bone biomaterials in support of sinus augmentation. Also, recombinant human bone morphogenetic protein-2 in an absorbable collagen sponge carrier (rhBMP-2/ACS) has been shown to support clinically relevant bone formation when used to augment the maxillary sinus. OBJECTIVES: To evaluate local bone formation/dental implant osseointegration following implantation of two particulate bone biomaterials using the mini-pig sinus augmentation model. METHODS: Nine adult Göttingen mini-pigs were used for evaluation of a biphasic ceramic (15%/85% HA/ß-TCP) and an allogeneic mineralized bone biomaterial. Treatments randomized to contralateral sinus sites included sham-surgery (control) and biomaterials. Two threaded dental implants (ø4.0 × 11.5 mm) were placed at each sinus site. The animals were euthanized at 8 weeks for histologic analysis. RESULTS: Execution of the surgical protocol and healing was unremarkable. Limited infraorbital swelling was observed until suture removal. The biphasic ceramic and allogeneic bone biomaterials produced significantly increased bone formation (5.2 ± 1.9 mm and 4.9 ± 1.6 mm vs. 2.6 ± 0.5 mm, p < 0.05) and osseointegration (18.0 ± 6.0% and 25.1 ± 18.2% vs. 10.1 ± 8.0%, p < 0.05) over the sham-surgery control. No significant differences were observed between biomaterials. CONCLUSIONS: Implantation of biphasic ceramic or allogeneic bone biomaterials enhances bone formation in the mini-pig maxillary sinus, however, dental implant bone support is incomplete resulting in overall limited osseointegration.

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.

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.

Pre-clinical evaluation of a minimally invasive laryngeal pacemaker system in mini-pig.

Microlaryngoscopic enlargement techniques have been the standard treatment for bilateral vocal fold paralysis (BVFP) for decades. Laryngeal pacing is a promising alternative treatment based on the electrostimulation of the posterior cricoarytenoid (PCA) muscle. This paper reports on the results of a pre-clinical study aiming to evaluate this method. Eight Göttingen mini-pigs were implanted with a laryngeal pacemaker (LP) implant prototype and with two LP electrodes, one in each PCA muscle. The 6-week follow-up included endoscopic stimulation controls in general anaesthesia and radiographic controls of electrode integrity and position stability. Stimulation parameters for optimal glottal opening were evaluated via videolaryngoscopy. Histopathology was performed upon conclusion of the study. 7/8 (87.5 %) animals were successfully implanted with the LP implant prototype and two LP electrodes. In general, stimulation was effectively delivered and correlated with the expected PCA muscle activation. 2/14 (14.3 %) electrodes dislocated and 1/14 (7.1 %) electrode tip broke. The LP system used in this experiment to induce vocal fold abduction by means of selective functional electrical stimulation of the PCA showed promising results. It may be a valid alternative to the current golden standard for BVFP treatment. Clinical studies are needed to confirm the medical relevance of the LP.

Use of quantitative MRI for the detection of progressive cartilage degeneration in a mini-pig model of osteoarthritis caused by anterior cruciate ligament transection.

BACKGROUND: To investigate the progression of cartilage degeneration using delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) and T2 mapping in a mini-pig model of osteoarthritis (OA) caused by anterior cruciate ligament transection (ACLT). METHODS: Twelve mini-pigs underwent ACLT in the left knee and were monitored by dGEMRIC (T1, gadolinium [Gd]) and T2 mapping after 2, 4, or 6 weeks (n = 4 each). No ACLT surgery was performed in four healthy control mini-pigs, which were also monitored by dGEMRIC and T2 mapping. Cartilage samples from the weight-bearing regions of the left medial femoral condyles were collected for macroscopic, histological, immunohistochemical, and biochemical analysis. Correlations between biochemical contents and T1,Gd and T2 values were evaluated using Pearson correlation analysis. RESULTS: T1,Gd values were gradually reduced and T2 values increased over time. Cartilage surfaces showed roughness at 4 weeks and additional defects at 6 weeks. Glycosaminoglycan (GAG) distribution and content gradually reduced over time (P < 0.05), and collagen distribution and anisotropy were obviously changed at 6 weeks. However, collagen content did not differ significantly among postoperative timepoints. GAG content was positively related to T1,Gd values (r = 0.888; P < 0.001) and negatively related to T2 values (r = -0.865; P < 0.001). Collagen content had no discernible correlation with T1,Gd or T2 values. CONCLUSION: dGEMRIC and T2 mapping can monitor the progression of cartilage degeneration in a mini-pig model of ACLT-induced OA, permitting early detection of OA.

Overexpression of Banna mini-pig inbred line fatty acid binding protein 3 promotes adipogenesis in 3T3-L1 preadipocytes.

Fatty acid binding protein 3 (H-FABP, FABP3) has been significantly associated with intramuscular fat (IMF) content in pigs, which is positively correlated with palatability of pork. However, its underlying function is not fully elucidated. We have investigated the effects of overexpression of the FABP3 gene on differentiation and adipogenesis of 3T3-L1 preadipocytes in the fat Banna mini-pig inbred line (fBMIL). Eukaryotic vectors that expressed the FABP3 protein were constructed, and stably established in the 3T3-L1 preadipocytes cell line. Cells were induced in a standard differentiation cocktail. Morphological changes and the degree of adipogenesis were measured by Oil Red O staining assay and triacylglycerol content measurement, respectively. mRNA expression levels of triacylglycerol metabolism-related genes were measured by qPCR. FABP3 significantly promoted differentiation of 3T3-L1 cells and enhanced triacylglycerol levels (P < 0.05). mRNA of the peroxisome proliferator-activated receptor γ (PPARγ), adipocyte fatty acid binding protein (422/aP2) and glycerol-3-phosphate dehydrogenase (GPDH) gene increased markedly (P < 0.05). In conclusion, expression of the FABP3 gene enhances adipogenesis in 3T3-L1 preadipocytes primarily by upregulating lipogenic PPARγ, 422/aP2 and GPDH genes.

Crude protein content in diets associated with intestinal microbiome and metabolome alteration in Huanjiang mini-pigs during different growth stages.

Introduction: Adequate crude protein (CP) content in diets plays a crucial role in the intestinal health of the animal. This study investigated the impacts of CP content in diets on the intestinal microbiome and metabolome profiles in growing Huanjiang mini-pigs. Methods: A total of 360 pigs with similar body weight (BW) were allocated for three independent feeding trials based on three different BW stages, including (i) 5-10 kg BW, diets consisting of 14, 16, 18, 20, and 22% CP content; (ii) 10-20 kg BW, diets consisting of 12, 14, 16, 18, and 20% CP content; and (iii) 20-30 kg BW, diets consisting of 10, 12, 14, 16, and 18% CP content. These experiments lasted 28, 28, and 26 days, respectively. Results: The results showed that the Shannon and Simpson indices were decreased (p < 0.05) in the ileum of pigs in response to the 14-18% CP compared with the 20% CP content at 5-10 kg BW stage, while diets containing 12 and 14% CP had higher Chao1 (p < 0.05) and Shannon (p = 0.054) indices compared with 18% CP at 20-30 kg BW stage. Compared with the 20% CP, the diet containing 16% CP displayed an increasing trend (p = 0.089) of Firmicutes abundance but had decreased (p = 0.056) Actinobacteria abundance in the jejunum at 5-10 kg BW stage. In addition, a diet containing 16% CP had higher Lactobacillus abundance in the jejunum and ileum compared with the 18, 20, and 22% CP, while had lower Sphingomonas and Pelomonas abundances in the jejunum and Streptococcus abundance in the ileum compared with the diet containing 22% CP (p < 0.05). Diets containing lower CP content altered differential metabolites in the small intestine at the early stage, while higher CP content had less impact. Conclusion: These findings suggest that a diet containing lower CP content (16% CP) may be an appropriate dietary CP content for 5-10 kg Huanjiang mini-pigs, as 16% CP content in diet has shown beneficial impacts on the intestinal microbiome and metabolome profiles at the early growth stage of pigs.

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.

Molecular characteristics and transcriptional regulatory of spermatogenesis-related gene RFX2 in adult Banna mini-pig inbred line (BMI).

RFX2 plays critical roles in mammalian spermatogenesis and cilium maturation. Here, the testes of 12-month-old adult boars of Banna mini-pig inbred line (BMI) were subjected to whole-transcriptome sequencing. The results indicated that the average expression (raw count) of RFX2 gene in BMI testes was 16138.25, and the average expression value of the corresponding transcript ENSSSCT00000043271.2 was 123.1898. The CDS of RFX2 obtained from BMI testes was 2,817 bp (GenBank accession number: OL362242). Gene structure analysis showed that RFX2 was located on chromosome 2 of the pig genome with 19 exons. Protein structure analysis indicated that RFX2 contains 728 amino acids with two conserved domains. Phylogenetic analysis revealed that RFX2 was highly conserved with evolutionary homologies among mammalian species. Other analyses, including PPI networks, KEGG, and GO, indicated that BMI RFX2 had interactions with 43 proteins involving various functions, such as in cell cycle, spermatid development, spermatid differentiation, cilium assembly, and cilium organization, etc. Correlation analysis between these proteins and the transcriptome data implied that RFX2 was significantly associated with FOXJ1, DNAH9, TMEM138, E2F7, and ATR, and particularly showed the highest correlation with ATR, demonstrating the importance of RFX2 and ART in spermatogenesis. Functional annotation implied that RFX2 was involved in 17 GO terms, including three cellular components (CC), six molecular functions (MF), and eight biological processes (BP). The analysis of miRNA-gene targeting indicated that BMI RFX2 was mainly regulated by two miRNAs, among which four lncRNAs and five lncRNAs competitively bound ssc-miR-365-5p and ssc-miR-744 with RFX2, respectively. Further, the dual-luciferase report assay indicated that the ssc-miR-365-5p and ssc-miR-744 significantly reduced luciferase activity of RFX2 3'UTR in the 293T cells, suggesting that these two miRNAs regulated the expression of RFX2. Our results revealed the important role of RFX2 in BMI spermatogenesis, making it an intriguing candidate for follow-up studies.