Wuzhishan Pigs Research Articles

N-glycosylation Modification Reveals Insights into the Oxidative Reactions of Liver in Wuzhishan Pigs

Although porcine liver contributes to their growth and development by nutrition production and energy supply, oxidative stress-induced hepatocyte damage is inevitable during metabolism. N-glycosylation is a common modification in oxidation; nevertheless, the effects of N-glycosylation on pig liver oxidative reactions remain undefined. In this study, liver proteins with N-glycosylation were detected in Wuzhishan (WZS) pigs between 4 and 8 months old and Large White (LW) pigs at 4 months old based on LC-MS/MS. The results showed that the number of differentially expressed proteins (DEPs) was larger between different pig cultivars than that between WZS pigs at various growth periods. The enriched pathways of DEPs were mainly related to oxidative reactions, and 10 proteins were finally selected that primarily consisted of CYPs, GSTs and HSPs with expressions significantly correlating to liver size and weight. The oxidative genes shared N-glycosylation-modified models of N-x-S and N-G. Five out of 10 proteins were upregulated in WZS pigs compared to LW pigs at 4 months old, while five proteins increased in WZS pigs from 4 to 8 months old. In conclusion, this research provides valuable information on the N-glycosylation motifs in liver oxidation genes of WZS pigs.

Polyurethane foam dressing with non-adherent membrane improves negative pressure wound therapy in pigs.

Negative pressure wound therapy (NPWT) is considered to be an effective technique to promote the healing of various wounds. The aim of this study was to evaluate different wound dressings combined with NPWT in treating wounds in Wuzhishan pigs. Excisions were made in the backs of the pigs and were covered with polyvinyl alcohol (PVA) dressing, polyurethane (PU) dressing or PU dressing with non-adherent membrane (PU-non-ad). NPWT was applied to the wound site. In the control group, basic occlusive dressing (gauze) without NPWT was applied. On days 0, 3, 7, 14, 21 and 28 post-surgery, the wound size was measured during dressing change, and wound healing rate (WHR) was calculated. In addition, blood perfusion within 2cm of the surrounding wound was measured by laser doppler flowmetry. Dressing specimen was collected and microbiology was analysed. Granulation tissues from the central part of the wounds were analysed for histology, vascular endothelial growth factor (VEGF) and cluster of differentiation 31 (CD31) mRNA expression. The PU-non-ad-NPWT significantly (p<0.01) accelerated wound healing in the pigs. Further pathological analysis revealed that the non-adherent membrane effectively protected granulation tissue formation in PU-NPWT treated wounds. The blood perfusion analysis suggested that the non-adherent membrane improved the blood supply to the wound area. Microbiological analysis showed that non-adherent membrane decreased the bacterial load in the PU-NPWT dressing. VEGF and CD31 mRNA expression was upregulated in the wound tissue from the PU-non-ad-NPWT treated groups. In this study, the PU dressing with non-adherent membrane was an ideal dressing in NPWT-assisted wound healing.

The Genetic Selection of HSPD1 and HSPE1 Reduce Inflammation of Liver and Spleen While Restraining the Growth and Development of Skeletal Muscle in Wuzhishan Pigs.

Wuzhishan (WZS) pigs, which are minipigs native to Hainan Province in China, are characterized by strong resistance to extreme hot temperatures and humidity. The relationship between their immune response and growth still needs to be clarified. In this study, we used whole genome sequencing (WGS) to detect variations within 37 WZS pigs, 32 Large White (LW) pigs, and 22 Xiangxi black (XXB) pigs, and ~2.49 GB of SNPs were obtained. These data were combined with those of two other pig breeds, and it was found that most of the genes detected (354) were located within the distinct genetic regions between WZS pigs and LW pigs. The network that was constructed using these genes represented a center including 12 hub genes, five of which had structural variations (SVs) within their regulatory regions. Furthermore, RNA-seq and RT-qPCR data for 12 genes were primarily consistent in liver, spleen, and LDM tissues. Notably, the expression of HSPs (HSPD1 and HSPE1) was higher while that of most genes involved in the JAK3-STAT pathway were lower in liver tissue of WZS pigs, compared with LW pigs. This likely not only reduced inflammation-related immune response but also impaired their growth. Our findings demonstrated the role of HSPs in the connection between inflammation and growth rate, while also providing the fundamental genetic selection of the adaptability of WZS pigs.

Integrated Analysis of Transcriptome Expression Profiles Reveals miRNA-326-NKX3.2-Regulated Porcine Chondrocyte Differentiation.

The porcine body length trait is an essential factor affecting meat production and reproductive performance. It is evident that the development/lengthening of individual vertebrae is one of the main reasons for increases in body length; however, the underlying molecular mechanism remains unclear. In this study, RNA-seq analysis was used to profile the transcriptome (lncRNA, mRNA, and miRNA) of the thoracic intervertebral cartilage (TIC) at two time points (1 and 4 months) during vertebral column development in Yorkshire (Y) and Wuzhishan pigs (W). There were four groups: 1- (Y1) and 4-month-old (Y4) Yorkshire pigs and 1- (W1) and 4-month-old (W4) Wuzhishan pigs. In total, 161, 275, 86, and 126 differentially expressed (DE) lncRNAs, 1478, 2643, 404, and 750 DE genes (DEGs), and 74,51, 34, and 23 DE miRNAs (DE miRNAs) were identified in the Y4 vs. Y1, W4 vs. W1, Y4 vs. W4, and Y1 vs. W1 comparisons, respectively. Functional analysis of these DE transcripts (DETs) demonstrated that they had participated in various biological processes, such as cellular component organization or biogenesis, the developmental process, the metabolic process, bone development, and cartilage development. The crucial bone development-related candidate genes NK3 Homeobox 2 (NKX3.2), Wnt ligand secretion mediator (WLS), gremlin 1 (GREM1), fibroblast growth factor receptor 3 (FGFR3), hematopoietically expressed homeobox (HHEX), (collagen type XI alpha 1 chain (COL11A1), and Wnt Family Member 16 (WNT16)) were further identified by functional analysis. Moreover, lncRNA, miRNA, and gene interaction networks were constructed; a total of 55 lncRNAs, 6 miRNAs, and 7 genes formed lncRNA-gene, miRNA-gene, and lncRNA-miRNA-gene pairs, respectively. The aim was to demonstrate that coding and non-coding genes may co-regulate porcine spine development through interaction networks. NKX3.2 was identified as being specifically expressed in cartilage tissues, and it delayed chondrocyte differentiation. miRNA-326 regulated chondrocyte differentiation by targeting NKX3.2. The present study provides the first non-coding RNA and gene expression profiles in the porcine TIC, constructs the lncRNA-miRNA-gene interaction networks, and confirms the function of NKX3.2 in vertebral column development. These findings contribute to the understanding of the potential molecular mechanisms regulating pig vertebral column development. They expand our knowledge about the differences in body length between different pig species and provide a foundation for future studies.

Novel Haplotype in the HHEX Gene Promoter Associated with Body Length in Pigs.

The screening of important candidate genes and the identification of genetic markers are important for molecular selection in the pig industry. The hematopoietically expressed homeobox (HHEX) gene plays an important role in embryonic development and organogenesis; however, the genetic variation and expression pattern of the porcine HHEX gene remains to be clarified. In this study, semiquantitative RT-PCR and immunohistochemistry results showed the specific expression of the HHEX gene in porcine cartilage tissues. A novel haplotype consisting of two SNPs rs80901185 (T > C) and rs80934526 (A > G) was detected in the promoter region of the HHEX gene. The expression of the HHEX gene was significantly higher in Yorkshire pigs (TA haplotype) than in Wuzhishan pigs (CG haplotype), and a population analysis showed that this haplotype was significantly associated with body length. An analysis subsequently revealed that the -586 to -1 bp region of the HHEX gene promoter showed the highest activity. Furthermore, we found that the activity of the TA haplotype was significantly higher than that of the CG haplotype by changing the potential binding of transcription factors YY1 and HDAC2. In summary, we conclude that the porcine HHEX gene may contribute to the breeding of pigs for body length traits.

Transmission of the gut microbiome in cohousing goats and pigs.

Social interaction facilitates the horizontal transmission of the microbiota between different individuals. However, little is known about the level of microbiota transmission in different livestock animals and different digestive tracts. The Hainan black goat and Wuzhishan pig are typical tropical local breeds on Hainan Island in China. Thus, we sampled and analyzed the gut microbiome in Hainan black goats (cecum and rumen) and Wuzhishan pigs (cecum) to study horizontal transmission by rearing them in the same pen (six goats and six pigs) or separate pens (nine goats and nine pigs). De novo assembly and binning recovered 3,262 strain-level and 2,488 species-level metagenome-assembled genomes (MAGs) using ∼1.3 Tb sequencing data. Of these MAGs, 1,856 MAGs were identified as novel strain. Compared with goats living in separate pens, social interaction in the same pen promotes community homogeneity in the rumen microbiome (P < 0.05) and the cecum microbiome (P < 0.05), respectively. Notably, approximately 7.08% (231/3262) of the gut microbial population could transmit during cohousing, 12 strains only in inter-species transmission, versus 190 strains only in intra-species transmission, and 10 strains only in foregut and hindgut transmission. In addition, the social contact group has high transmitted strain abundance, which is correlated with community composition. This study provided a new insight into the influence of social interaction on the animal gut microbiota.

Porcine Endogenous Retroviruses: Quantification of the Viral Copy Number for the Four Miniature Pig Breeds in China.

Domestic pigs has served not only as one of the most important economy livestock but also as ideal organ-source animals owing to similarity in anatomy, physiology, and organ size to humans. Howerer, the barrier of the cross-species transmission risk of porcine endogenous retrovirus (PERVs) blocked the pig-to-human xenotransplantation. PERVs are integrated into pigs’ genomes and cannot be eliminated by designated or specified pathogen-free breeding. PERVs are an important biosafety issue in xenotransplantation because they can be released from normal pig cells and infect human cells in vitro under certain conditions. Screening and analyzing the presence of PERVs in pig genome will provide essential parameters for pig breed sources. In China, four miniature pig breeds, such as Guizhou miniature pig (GZ), Bama miniature pig (BM), Wuzhishan miniature pig (WZS), and Juema miniature pig (JM), were the main experimental miniature pig breeds, which were widely used. In this study, PCR was performed to amplify env-A, env-B, and env-C for all individuals from the four breeds. The results revealed that PERV env-A and env-B were detected in all individuals and the lowest ratios of PERV env-C was 17.6% (3/17) in the GZ breed. Then, PERV pol and GAPDH were detected using the droplet digital PCR (ddPCR) method. As the reference of GAPDH copy number, the copy numbers of PERVs were at the median of 12, 16, 14, and 16 in the four miniature pig breeds (GZ, BM, WZS, and JM), respectively. Furthermore, the copy number of the PERV pol gene in many organs from the GZ breed was analyzed using ddPCR. The copy numbers of PERV pol gene were at the median of 7 copies, 8 copies, 8 copies, 11 copies, 5 copies, 6 copies, and 7 copies in heart, liver, spleen, lung, kidney, muscle, and skin, and the maximum number was 11 copies in the lung. The minimum number was 5 copies in the kidney as the reference of GAPDH. These data suggest that GZ breed has the lower PERVs copy number in the genome, and may be an ideal organ-source miniature pig breed for the study of the pig-to-human xenotransplantation.

Fecal microbial composition and functional diversity of Wuzhishan pigs at different growth stages

The mammalian gut microbiome participates in almost all life processes in the host. In addition to diet, the breed is the main factor affecting changes in the swine gut microbiota. The composition of the gut microbiota changes significantly during different growth stages. Research on developmental changes in the gut microbiota of indigenous Chinese pig breeds is limited. In this study, the fecal microbiota of Wuzhishan pigs (a Chinese indigenous miniature pig) at different growth stages was investigated using high-throughput 16S rRNA sequencing. Firmicutes and Bacteroidetes were the two dominant phyla, accounting for more than 80% of all sequences. With increasing age, the fecal microbial diversity increased, and the proportion of Firmicutes increased, whereas the proportion of Bacteroidetes decreased. A total of 49 biomarkers with statistical differences were detected in the four growth stages. The different microbiota among groups enhanced the ability to degrade fiber, carbohydrates, and other substances during the growth stages. The endocrine system was different in multiple growth stage paired comparisons, which was attributed to the different body statuses in the growth stages. This study revealed developmental changes in the structure and function of gut microbes in local pigs.

Effect of GM maize on metabolism and performance of chicken

Effect of GM maize on metabolism and performance of chicken

Long-term case study of a Wuzhishan miniature pig with diabetes.

BackgroundMiniature pigs are attractive animal models for exploring diabetes because they are similar to humans in terms of physiological structure and metabolism. However, little is known about the complications of diabetes in pigs.MethodsIn this study, a 28‐month observation of a Wuzhishan miniature pig with streptozotocin (STZ)‐induced (120 mg/kg) diabetes was conducted, to investigate diabetes‐related complications and the possibility of self‐recovery in miniature pigs. Blood glucose, serum and urinary biochemistry was measured, and histopathologic examinations of eyes, kidney and pancreas were made.ResultsDuring the observation, diabetic complications of eyes and kidney were observed. The eye complications included bilateral cataracts in the 15th month and degeneration of inner retina and microaneurysm in the 28th month. Kidney complications included glomerular mesangial expansion, focal segmental glomerular sclerosis, and renal tubular epithelial degeneration, but no proteinuria was observed. By 28 months after the application of STZ, with no treatment given, blood glucose had recovered and the number of pancreatic islet beta‐cells had increased significantly.ConclusionsWe showed that the STZ‐induced diabetes model in miniature pigs could accurately mimic the pathological changes of human diabetes, and that pancreatic islet beta‐cell regeneration did occur in an adult miniature pig, providing a new means for exploring diabetic complications and pancreatic islet beta‐cell regeneration.

Effect of butylphthalide injection to mitochondrial function of porcine cerebral neuron after cardiopulmonary resuscitation

Objective To investigate the mechanism of cerebral protection by treatment of butylphthalide (NBP) and its effect to mitochondria in a porcine model of cardiac arrest (CA) after cardiopulmonary resuscitation (CPR). Methods Healthy Wuzhishan pigs weighting (30±2) kg were divide into three groups randomly(random number): The sham group (n=6), the control group (n=12) and the NBP group (n=12). Operation was performed in the sham group. Cardiac arrest of ventricular fibrillation was induced by programed electrical stimulation in the control and NBP group. After CPR, asynchronous defibrillation of 150J was performed to achieve the restoration of spontaneous circulation. NBP was injected at the rate of 2.5 mg•kg-1 in the NBP group. Hemodynamics were recorded at baseline, 1 hr, 2 hr, 3 hr and 4 hr after CPR. The number of injured neurons, apoptosis index and evaluation of mitochondrial injury were calculated under light and electrical microscope respectively. Mitochondria were separated by differential centrifugation. Mitochondrial respiratory function was measured with oxygen consumption of R3 and R4, respiratory control rate (RCR), ADP/oxygen. Mitochondrial permeability transition pore (MPTP) open was tested by colorimetric. Results After CPR, the mean artery pressure, coronary perfusion pressure and cardiac output decreased significantly, whereas no significant differences were found between the control and NBP group (P>0.05). Significant cerebral injury was found after CPR. The number of injured neurons, apoptosis index and evaluation of mitochondrial injury were improved significantly by the NBP treatment (P<0.05). Compared with the sham group, oxygen consumption of R3 and R4, R3/R4 and ADP/O decreased significantly in the cerebral frontal cortex mitochondria of the control group (P<0.01), whereas they were increased in the NBP group (P<0.01). MPTP increased in the control group, which could be improved by the NBP treatment. Conclusions NBP can improve the neurologic outcome after CPR and decrease the apoptosis of neurons by improving the respiratory function of mitochondria and inhibiting the MPTP opening. Key words: Cardiac arrest; Cardiopulmonary resuscitation; Butylphthalide injection; Neurologic outcome; Mitochondrial function

Genomic Analysis Reveals Specific Patterns of Homozygosity and Heterozygosity in Inbred Pigs.

Simple SummaryThe inbred strain of miniature pig is an ideal model for biomedical research due to its high level of homozygosity. Our results from multidimensional scaling, admixture, and phylogenetic analyses indicated that the inbred Wuzhishan pig (WZSP), with its unique genetic properties, can be utilized as a novel genetic resource for pig genome studies. Inbreeding depression and run of homozygosity (ROH) analyses revealed an average of 61 and 12 ROH regions in the inbred and non-inbred genomes of WZSPs, respectively. ROH number, length, and distribution across generations indicate the impacts of recombination and demography on ROHs in these WZSPs. Finally, we detected 56 single nucleotide polymorphisms (SNPs) showing constant heterozygosity with heterozygosity (He) = 1 across six generations in inbred pigs, while only one was found in the non-inbred population. Among these SNPs, we observed nine SNPs located in swine RefSeq genes, which were found to be involved in signaling and immune processes. Together, our findings indicate that the inbred-specific pattern of homozygosity and heterozygosity in inbred pigs can offer valuable insights for elucidating the mechanisms of inbreeding in farm animals.The inbred strain of miniature pig is an ideal model for biomedical research due to its high level of homozygosity. In this study, we investigated genetic diversity, relatedness, homozygosity, and heterozygosity using the Porcine SNP60K BeadChip in both inbred and non-inbred Wuzhishan pigs (WZSPs). Our results from multidimensional scaling, admixture, and phylogenetic analyses indicated that the inbred WZSP, with its unique genetic properties, can be utilized as a novel genetic resource for pig genome studies. Inbreeding depression and run of homozygosity (ROH) analyses revealed an average of 61 and 12 ROH regions in the inbred and non-inbred genomes of WZSPs, respectively. By investigating ROH number, length, and distribution across generations, we further briefly studied the impacts of recombination and demography on ROH in these WZSPs. Finally, we explored the SNPs with higher heterozygosity across generations and their potential functional implications in the inbred WZSP. We detected 56 SNPs showing constant heterozygosity with He = 1 across six generations in inbred pigs, while only one was found in the non-inbred population. Among these SNPs, we observed nine SNPs located in swine RefSeq genes, which were found to be involved in signaling and immune processes. Together, our findings indicate that the inbred-specific pattern of homozygosity and heterozygosity in inbred pigs can offer valuable insights for elucidating the mechanisms of inbreeding in farm animals.

Transcriptomic Analysis of Coding Genes and Non-Coding RNAs Reveals Complex Regulatory Networks Underlying the Black Back and White Belly Coat Phenotype in Chinese Wuzhishan Pigs.

Coat color is one of the most important characteristics for distinguishing Chinese indigenous pig breeds. In Wuzhishan pigs, the animals have black on the back and white on the abdomen. However, the molecular genetic basis of this phenotype is unclear. In this study, we used high-throughput RNA sequencing to compare expression profiles of coding and non-coding RNAs from white and black skin samples obtained from individual Wuzhishan pigs. The expression profiling revealed that 194 lncRNAs (long non-coding RNAs), 189 mRNAs (messenger RNAs), and 162 miRNAs (microRNAs) had significantly different levels of expression (|log2 fold change| > 1, p-value < 0.05) in white and black skin. Compared to RNA levels in black skin, white skin had higher levels of expression of 185 lncRNAs, 181 mRNAs, and 23 miRNAs and lower levels of expression of 9 lncRNAs, 8 mRNAs, and 139 miRNAs. Functional analysis suggested that the differentially expressed transcripts are involved in biological processes such as melanin biosynthesis, pigmentation and tyrosine metabolism. Several key genes involved in melanogenesis, including MLANA, PMEL, TYR, TYRP1, DTC, TRPM1 and CAMK2A, had significantly different levels of expression in the two skin tissues. Potential lncRNA–miRNA–gene interactions were also examined. A total of 15 lncRNAs, 11 miRNAs and 7 genes formed 23 lncRNA–miRNA–gene pairs, suggesting that complex regulatory networks of coding and non-coding genes underlie the coat color trait in Wuzhishan pigs. Our study provides a foundation for understanding how lncRNA, miRNA and genes interact to regulate coat color in black-back/white-belly pigs. We also constructed lncRNA–miRNA–gene interaction networks to elucidate the complex molecular mechanisms underlying skin physiology and melanogenesis. The results extend our knowledge about the diversity of coat color among different domestic animals and provide a foundation for studying novel mechanisms that control coat color in Chinese indigenous pigs.

Effects of Transgenic Bt Rice Containing the Cry1Ab Protein on the Gastrointestinal Health of Highly Inbred Wuzhishan Pigs after Two Generations of Feeding.

To assess the safety of transgenic Bt rice, we evaluated the chronic effects of Bt rice carrying the Cry1Ab protein (1.64 mg/kg) on the gut microbiota and histopathology in highly inbred Wuzhishan pigs (WZSP) in a two-generation feeding study. The WZSP of the f0 ( n = 26) and f1 ( n = 27) generations were divided into GM and Non-GM groups and fed for 360 and 420 days with Bt and isogenic parent-line rice, respectively. The high-throughput 16S rRNA gene sequencing results revealed that the relative abundance of the family Enterobacteriaceae and genus Escherichia were significantly different between f1F and f1Z. The level of the genera CF231 and SMB53 in the f0Z group was higher than that in the f0F group. In addition, no abnormal histopathology changes in the main gastrointestinal tissues were found after feeding with Bt rice. In conclusion, Bt rice consumption has no adverse effects on the gut microbiota or organ function of WZSP.

Dynamic imaging and pathological changes in pig liver after MR-guided microwave ablation

BackgroundMagnetic resonance (MR)-guided microwave ablation is a well-developed technique for the treatment of tumors, especially hepatic carcinomas. However, there are no detailed reports on the changes in the MR images and histology observed after the ablation. This study aimed to dynamically map the pathological changes after ablation and the changes occurring on MR images.MethodsWe performed MR-guided microwave ablation in 10 Wuzhishan pigs and obtained an MR scan immediately after ablation (0 weeks) and at 1, 2, 3, and 4 weeks after ablation. We compared the ablation assessed on MR images to tissue specimens obtained during follow-up.ResultsWe found no significant difference in the ablation size between MR images and tissue specimens; the mean length and width of the ablated zone were 4.27 cm and 2.42 cm, respectively, on MR images and 4.26 cm and 2.45 cm, respectively, on specimens (P > 0.05). Immediately after ablation, carbonization and cavities were observed in the center of the ablation zone. Surrounding layer cells were necrotic but maintained their original shapes. The outermost layer was inflamed, but gradually showed fibrotic characteristics. The MR images accurately reflected the exact histological tissue changes after the ablation procedure.ConclusionThe dynamic imaging and pathological features of liver ablation outlined in this study will provide a useful reference for patient follow-up after MR-guided microwave ablation.

Effects of long-term feeding with genetically modified Bt rice on the growth and reproductive performance in highly inbred Wuzhishan pigs

This study aimed to evaluate the safety of GM rice containing Cry1Ab protein (1.64 mg/kg) as the main ingredient in the diet after 360 days of feeding in the highly inbred Wuzhishan pigs (WZSP). A total of 28 pigs aged 3 months were divided into two groups and fed with either non-GM (MH86 group, female n = 7, male n = 7) or GM rice (Bt group, female n = 7, male n = 7). The total dosage of Cry1Ab protein for female and male pigs was 0.972 and 0.963 mg/kg body weight by the end of final weighing date (D 75), respectively. The results showed that there were no differences in the average daily gain, average daily food intake, body weight, feed conversion ratio, oestrus cycle duration, oestrus period, litter size, serum steroids, or the birth and weaning weight of offspring between Bt and MH86 groups. All pigs (n = 28) were sacrificed to collect tissues and blood samples for the analysis of relative organ weights, blood physiological and biochemical parameters and histopathological examinations. No significant differences existed in the tested indicators except that total protein was significantly higher in the MH86 compared to the Bt group (P < 0.05). In addition, the total bilirubin in the MH86 group was higher than the Bt group for females (P < 0.05), but no difference existed in males (P > 0.05). No pathologic abnormal changes were found from histopathological examinations of the main tissues. On the basis of these results, feeding with Bt rice for 360 days did not affect the growth, reproductive performance, hematology, or organ morphology in WZSP.

Rapid and low-cost strategy for detecting genome-editing induced deletion: A single-copy case

Genome editing techniques have been implemented in human daily lives, which has created a high demand for the development of new gene-edited product analysis methods. Conventional assays are time-consuming, labor-intensive, and costly. This paper proposes a rapid and low-cost strategy for detecting genome-editing induced deletion which works by integrating rapid-multiplex ligation-dependent probe amplification (MLPA) with a dual-lateral flow nucleic acid biosensor (LFNAB) cascade in a single-copy case. A rapid-MLPA was first introduced to the LFNAB system as a replacement for the conventional PCR for enhanced specificity and accuracy. A dual-LFNAB was applied for the detection of genome-editing induced deletion without any additional instrumentation or complex operation. After optimization, we achieved the specific detection of wildtype alleles and deletion alleles in spiked samples with a detection limit of 0.4 fM, which is comparable to that of electrophoresis-based detection assays and fluorescent biosensors. To confirm the validity and feasibility of our strategy, we assayed two pork samples from two WUZHISHAN pigs successfully. By comparing the detection results from next-generation sequencing analysis, we found that the proposed cascade demonstrates at least 20-fold shorter assay time and at least 100-fold less assay cost. To this effect, the proposed method is a rapid and low-cost solution to sample-to-answer detection of genome-editing induced deletion and shows remarkable potential in regards to international trade, transparency, and freedom of choice.

Полиморфизм тРНКLeu и тРНКSer митохондриальной ДНК свиней различных пород

Analysis of the sequence of mitochondrial DNA (mtDNA), which has a maternal nature of inheritance, is an effective way of assessing the individual characteristics of commercial lines. The analysis of the sequence variability of the mtDNA tRNA Ser and tRNA Leu genes in different pig breeds was carried out. The mtDNA tRNA Leu1 , tRNA Leu2 , tRNA Ser1 , tRNA Ser2 genes were chosen as the study objects. For the study of the sequences, data on pigs of different breeds were selected from the National Center for Biotechnological Information (NCBI). The sample group included 20 animals. Alignment of nucleotide sequences was performed using the BioEdit program. Sequence analysis showed the presence of polymorphisms in all studied genes. A total of six polymorphic sites are established, which are represented by transitions. Analysis of the gene tRNA eu showed the presence of A3892G substitution. The polymorphism A3892G in the Wuzhishan and Luchuan pigs was determined in the tRNA Leu1 gene, and polymorphism C3907T was determined in the Swedish wild boar. Many of the pigs studied had a simultaneous presence of two A12879G and C12883T transitions in the tRNA Leu2 gene. One transition of A8103G in the tRNA Ser1 gene was established in Large White pigs of Korean breeding. The polymorphism of the tRNA Ser2 gene is represented by the translation of T12838C into the Italian wild boar. The highest number of polymorphisms was determined in Large White of Korean and Chinese breeding, as well as in the Italian wild boar. The study of mtDNA polymorphism will allow to establish associative links and develop mtDNA markers that can be used in programs for improving and creating breeding resources in pig breeding.

Corneal Xenotransplantation From Pig to Rhesus Monkey: No Signs of Transmission of Endogenous Porcine Retroviruses

BackgroundXenotransplantations of pig corneas have become an attractive alternative to human corneas. Such xenotransplantations carry the danger, however, of transmission of porcine endogenous retroviruses (PERVs). Here, we investigated whether porcine corneas harbor viral DNA and RNA and whether transplantation to a nonhuman primate would lead to host PERV infection. MethodsMonkey vein endothelial cells (MVECs) were inoculated with porcine aortic endothelial cell (PAEC) supernatants, and DNA and total RNA of MVECs were tested for PERV by polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) assays. Corneas were harvested from Wuzhishan miniature pigs, and the presence of PERV proviral DNA and RNA was analyzed by PCR and RT-PCR, respectively. Fresh or dehydrated corneas were then transplanted to rhesus monkeys, and PERV proviral DNA and RNA were analyzed in host peripheral blood lymphocytes at 6 and 24 months. Furthermore, the presence of PERV sequences was analyzed in the transplant at 24 months. ResultsPCR analysis showed PERV transfection from PAECs to MVECs in vitro. PCR and RT-PCR gave positive signals for PERV subtypes A and B, but not PERV-C, regardless of how the corneas were prepared. No evidence was found for PERV transmission to the host, and the transplant had lost its viral signal at the end of the 24-month period. ConclusionsRegardless of cornea preparation and storage, PERV transmission from pig to host could not be detected, despite that the transplant was initially PERV-positive. The use of the Wuzhishan miniature pig as the donor may be advantageous because it lacks PERV-C and hence potentially infectious A/C recombinants.

Repair of articular cartilage defects by autologous bone mesenchymal stem cells and allogeneic costal chondrocytes in the knee of Wuzhishan miniature pigs

To investigate the feasibility of construction of tissue engineered cartilage by co-culture of bone marrow mesenchymal stem cells (BMSCs) and costal chondrocytes (CCs), and to provide theoretical basis and experimental basis for clinical repair of articular cartilage defects by Wuzhishan miniature pig knee cartilage defects with co-cultured cells. Methods: Density gradient centrifugation method was used to isolate BMSCs from Wuzhishan miniature pig. The double enzyme digestion method was used to isolate CCs. The passage 3 generation of BMSCs and passage 2 generation of CCs were randomly divided into 3 groups: a co-culture group of BMSCs:CCs for 1:2 (Group A), a simple CCs (Group B), and a simple BMSCs (Group C). The cell growth curve was drawn, and the content of glycosaminoglycan (GAG) of external separation in chondrocytes was determined. The 12 Wuzhishan miniature pigs were randomly divided into a co-culture cells/collagen membrane experimental group, a collagen membrane control group and the blank group. In the co-culture cells/collagen membrane experimental group, the co-cultured cells/collagen membrane were implanted into the cartilage defects of the mandibular condyle; in the collagen membrane control group, only collagen membrane was implanted; while in the blank group, nothing was implanted. Six animals were sacrificed at 8 and 16 weeks after surgery respectively (2 animals in each group). General observation, cartilage histological score and histopathological examination were carried out. Results: The BMSCs and co-culture cells grew well. The biological activity of CCs was good. After 16 weeks of operation, the repair tissues in the co-cultured cells/collagen membrane experimental group showed hyaline cartilage features: smooth, flat, and integrated well with the surrounding cartilage and subchondral bone. The collagen membrane in the collagen membrane control group was fibrously repaired. Repair tissue gross score in the co-culture cells/collagen membrane experimental group was significantly better than that in the collagen membrane control group and the blank group (both P<0.05), but there was no significant difference between the collagen membrane control group and the blank group (P>0.05). Conclusion: BMSCs, CCs and co-cultured cells can function as the seed cells for cartilage tissue engineering, and the co-culture cells (BMSCs:CCs=1:2) possess more advantages; the short-term effect of co-culture cells with collagen membrane on repairing cartilage defects is satisfied.