Betaine hydrochloride addition in Bama mini-pig's diets during gestation and lactation enhances immunity and alters intestine microbiota of suckling piglets.

BACKGROUND: Maternal nutrition during gestation and lactation is essential for offspring's health. The present study aimed to investigate the effects of betaine hydrochloride addition to sow diets during gestation and lactation on suckling piglet's immunity and intestine microbiota composition. Forty Bama mini-pigs were randomly allocated into two groups and fed a basal diet (control group) and a basal diet supplemented with 3.50 kg ton-1 betaine hydrochloride (betaine group) from day 3 after mating to day 21 of lactation. After 21 days of the delivery, 12 suckling piglets from each group with similar body weight were selected for sample collection. RESULTS: The results showed that maternal betaine hydrochloride addition decreased (P < 0.05) the plasma levels of interleukin (IL)-1ß, IL-2, IL-6, and tumor necrosis factor-α in suckling piglets. Furthermore, dietary betaine hydrochloride addition in sow diets increased (P < 0.05) the villus height (VH) and VH to crypt depth ratio in the jejunum and ileum of suckling piglets. In the piglets' intestinal microbiota community, the relative abundances of Roseburia (P < 0.05) and Clostridium (P = 0.059) were lower in the betaine group compared to those in the control group. Moreover, betaine hydrochloride addition in sow diets decreased the colonic tyramine (P = 0.091) and skatole (P = 0.070) concentrations in suckling piglets. CONCLUSION: Betaine hydrochloride addition in sow diets enhanced the intestinal morphology, improved immunity, and altered intestinal microbiota of suckling piglets. These findings indicated that betaine hydrochloride addition in sow diets during gestation and lactation will impact suckling piglets' health. © 2021 Society of Chemical Industry.

Dietary nutrient levels alter the metabolism of arginine family amino acids in the conceptus of Huanjiang mini-pigs.

BACKGROUND: The arginine family amino acids (AFAAs) exert important roles in the metabolism, growth and development of the conceptus. However, to date, few studies have investigated the effects of maternal nutrient levels on the concentrations and metabolism of AFAAs in the conceptus. RESULTS: Compared to low nutrient diets, high nutrient diets increased (P < 0.05) the concentrations of citrulline and proline (Pro) in plasma; the concentrations of arginine, glutamine, Pro and ornithine (Orn) in the amniotic fluid; and the concentrations of all detected AFAAs in the allantoic fluid, which were most pronounced on day 45 of pregnancy. High nutrient diets upregulated (P < 0.05) mRNA expression of arginase I (Arg I), Pro oxidase and spermidine synthetase (SRM) in the fetal placenta, as well as Arg II, SRM and spermine synthetase (SMS) expression in the fetal liver (most pronounced on day 45 of pregnancy). The same effect was observed for mRNA expression of NO synthase and Orn aminotransferase (OAT), mainly on day 110 of pregnancy, and for mRNA expression of Arg I, Arg II, OAT, Orn decarboxylase and SMS throughout pregnancy. High nutrient diets upregulated (P < 0.05) mRNA expression of Y+ L-type amino acid transporter (LAT) and cationic amino acid transporter 1 (CAT1) in the fetal jejunum throughout pregnancy. Dietary treatments did not affect (P > 0.05) mRNA expression of Y+ LAT1, sodium-coupled neutral amino acid transporter 2 (SNAT2) and CAT1 in the fetal placenta, skeletal muscle and colon. CONCLUSION: High nutrient diets increased the concentration and transport of AFAAs in the mothers and conceptus, which likely improves growth and development of the conceptus. © 2018 Society of Chemical Industry.

Fetal Huanjiang mini-pigs exhibit differences in nutrient composition according to body weight and gestational period.

Low birth weight may negatively affect energy storage and nutrient metabolism, and impair fetal growth and development. We analyzed effects of body weight (BW) and gestational period on nutrient composition in fetal Huanjiang mini-pigs. Fetuses with the lowest BW (LBW), middle BW (MBW), and highest BW (HBW) were collected at days 45, 75, and 110 of gestation. Crude protein (CP), crude fat, amino acid (AA), and fatty acid (FA) concentrations were determined. The BW gain, carcass weight, fat percentage, and uterus weight of sows increased as gestation progressed, as did litter weight, average individual fetal weight, fetal body weight, and dry matter (DM). The concentrations of Ala, Arg, crude fat, Gly, Pro, Tyr, C14:0, C16:0, C16:1, C18:1n9c, C18:2n6c, C18:3n3, C18:3n6, C20:0, C20:3n6, saturated FA (SFA), and monounsaturated FA (MUFA) increased significantly as gestation progressed. The percentage of skeleton, and the ratio of the liver, lung, and stomach to BW decreased as gestation progressed. There were also significant reductions in the concentrations of CP, Asp, Glu, His, Ile, Leu, Lys, Phe, Ser, Thr, essential AA (EAA), acidic AA, C17:0, C20:4n6, C22:6n3, unsaturated FA (UFA), polyunsaturated FA (PUFA), n-3PUFA, n-6PUFA as gestation progressed, and reductions in EAA/total AA (TAA), PUFA/SFA, and n-3/n-6 PUFA. The LBW fetuses exhibited the lowest BW and crude fat, C14:0, C16:1, C17:0, C18:2n6c, and MUFA concentrations at days 75 and 110 of gestation. They also exhibited lower Tyr concentration at day 45 of gestation and lower Glu concentration at day 75 of gestation than HBW fetuses. These findings suggest that LBW fetuses exhibit lower amounts of crude fat and several FAs during mid-gestation and late-gestation, which may in turn affect adaptability, growth, and development.

Generation of chimeric minipigs by aggregating 4- to 8-cell-stage blastomeres from somatic cell nuclear transfer with the tracing of enhanced green fluorescent protein.

BACKGROUND: Blastocyst complementation is an important technique for generating chimeric organs in organ-deficient pigs, which holds great promise for solving the problem of a shortage of organs for human transplantation procedures. Porcine chimeras have been generated using embryonic germ cells, embryonic stem cells, and induced pluripotent stem cells; however, there are no authentic pluripotent stem cells for pigs. In previous studies, blastomeres from 4- to 8-cell-stage parthenogenetic embryos were able to generate chimeric fetuses efficiently, but the resulting fetuses did not produce live-born young. Here, we used early-stage embryos from somatic cell nuclear transfer (SCNT) to generate chimeric piglets by the aggregation method. Then, the distribution of chimerism in various tissues and organs was observed through the expression of enhanced green fluorescent protein (EGFP). METHODS: Initially, we determined whether 4- to 8- or 8- to 16-cell-stage embryos were more suitable to generate chimeric piglets. Chimeras were produced by aggregating two EGFP-tagged Wuzhishan minipig (WZSP) SCNT embryos and two Bama minipig (BMP) SCNT embryos. The chimeric piglets were identified by coat color and microsatellite and swine leukocyte antigen analyses. Moreover, the distribution of chimerism in various tissues and organs of the piglets was evaluated by EGFP expression. RESULTS: We found that more aggregated embryos were produced using 4- to 8-cell-stage embryos (157/657, 23.9%) than 8- to 16-cell-stage embryos (100/499, 20.0%). Thus, 4- to 8-cell-stage embryos were used for the generation of chimeras. The rate of blastocysts development after aggregating WZSP with BMP embryos was 50.6%. Transfer of 391 blastocysts developed from 4- to 8-cell-stage embryos to five recipients gave rise to 18 piglets, of which two (11.1%) were confirmed to be chimeric by their coat color and microsatellite examination of the skin. One of the chimeric piglets died at 35 days and was subsequently autopsied, whereas the other piglet was maintained for the following observations. The heart and kidneys of the dead piglet showed chimerism, whereas the spinal cord, stomach, pancreas, intestines, muscle, ovary, and brain had no chimerism. CONCLUSIONS: To our knowledge, this is the first report of porcine chimeras generated by aggregating 4- to 8-cell-stage blastomeres from SCNT. We detected chimerism only in the skin, heart, and kidneys. Collectively, these results indicate that aggregation using 4- to 8-cell-stage SCNT embryos offers a practical approach for producing chimeric minipigs. Furthermore, it also provides a potential platform for generating interspecific chimeras between pigs and non-human primates for xenotransplantation.

Suberoylanilide hydroxamic acid, a novel histone deacetylase inhibitor, improves the development and acetylation level of miniature porcine handmade cloning embryos.

Histone deacetylase inhibitors (HDACis) can change the histone acetylation and significantly enhance the developmental competence of the pre-implantation SCNT embryo. To select a proper histone deacetylase inhibitor to improve the success rate and potentially developmental ability of handmade cloning (HMC) embryos of miniature porcine, we compared the effect of two histone deacetylase inhibitors (SAHA vs. VPA) on HMC embryo development, their histone acetylation level and the expression level of relevant genes. The blastocyst rate and number of blastocyst cells of HMC embryos treated with SAHA (SAHA-HMC) or VPA (VPA-HMC) were significantly higher than those of control (Control-HMC), respectively, but there were no significant difference between SAHA-HMC and VPA-HMC groups. In addition, the acetylation level (AcH4K8) of Control-HMC and VPA-HMC embryos at the blastocyst stage, respectively, was significantly lower than that of in vitro fertilized (IVF) and SAHA-HMC embryos. However, the acetylation H4K8 of the blastocysts had no significant difference between SAHA-HMC and the IVF groups. The SAHA-HMC blastocysts indicated comparative expression levels of Oct4 and HDAC1 (histone deacetyltransferase gene) with those of IVF blastocysts. In contrast, the expression levels of Oct4 were lower and those of HDAC1 were higher in the VPA-HMC and Control-HMC blastocysts, respectively, compared to those of the IVF blastocysts. Our results demonstrated that the HMC embryos treated by SAHA could promote the pre-implantation development and increase the levels of histone H4K8 acetylation and the expression of the OCT4 gene, yet decrease the expression of the HDAC1 gene to the comparable level of the IVF embryos. Our results proved that SAHA may be a better histone deacetylase inhibitor for porcine HMC compared to VPA, and furthermore, it may indicate that SAHA can effectively correct the abnormal histone acetylation during the HMC embryo development and subsequently improve the full-term developmental potential of the HMC embryos after embryo transplantation.

A Comprehensive Study of Palate Development in Miniature Pig.

Palate development is an important morphogenetic event in facial development, including the fusion of the lateral and medial nasal portions of the frontonasal process and maxilla. Derailments of any of these events may result in cleft palate, the most frequent congenital craniofacial abnormality. Recent research has shown that the microanatomy of the miniature pig oral maxillofacial region is quite similar to that of humans, and the use of miniature pigs as a large animal model for dental and orofacial research is increasing. Little information is available, however, about the development of the miniature pig palate. Here, using histological and ultrastructural methods, we describe the developmental stages of the palate in miniature pigs. Sections from E26, E30, E35, E40, E45, and E50 embryos were stained with hematoxylin-eosin, and selected specimens were also processed for electron microscopy. The development of the miniature pig palate can be divided into four stages: growth of the bilateral palatal shelves alongside the tongue at E30; elevation of the horizontal position above the tongue at E35; establishment of bilateral shelf contact at the midline from E35-50; and a final fusion step at E50, similar to the mouse and human. The histological characteristics of the miniature pig palate at different developmental stages were synchronously verified at the ultrastructural level. Our study provides a piece of first-hand data regarding palate morphological organogenesis in the miniature pig and a foundation for further research with this model to explore mechanisms of cleft palate development. Anat Rec, 300:1409-1419, 2017. © 2017 Wiley Periodicals, Inc.

Gnotobiotic Miniature Pig Interbreed Somatic Cell Nuclear Transfer for Xenotransplantation.

Transgenic animal producing technology has improved consistently over the last couple of decades. Among the available methods, somatic cell nuclear transfer (SCNT) technology was officially the most popular. However, SCNT has low efficiency and requires a highly skilled individual. Additionally, the allo-SCNT nuclear reprogramming mechanism is poorly understood in the gnotobiotic miniature pig, which is a candidate for xenotransplantation, making sampling in oocytes very difficult compared to commercial hybrid pigs. Therefore, interbreed SCNT (ibSCNT), which is a combination of miniature pig and commercial pig (Landrace based), was analyzed and was found to be similar to SCNT in terms of the rate of blastocyst formation (12.6% ± 2.9% vs. 15.5% ± 2.2%; p > 0.05). However, a significantly lower fusion rate was observed in the ibSCNT compared to normal SCNT with Landrace pig somatic cells (29.6% ± 0.8% vs. 65.0% ± 4.9%). Thus, the optimization of fusion parameters was necessary for efficient SCNT. Our results further revealed that ibSCNT by the whole-cell intracytoplasmic injection (WCICI) method had a significantly higher blastocyst forming efficiency than the electrofusion method (31.1 ± 8.5 vs. 15.5% ± 2.2%). The nuclear remodeling and the pattern of changes in acetylation at H3K9 residue were similar in both SCNT and ibSCNT embryos.

iTRAQ-based quantitative proteomic analysis reveals the distinct early embryo myofiber type characteristics involved in landrace and miniature pig.

BACKGROUND: Pig (Sus scrofa) is a major source of dietary proteins for human consumption and is becoming a valuable model in agricultural and biomedical research. The recently developed isobaric tag for relative and absolute quantitation (iTRAQ) method allows sensitive and accurate protein quantification. Here, we performed the first iTRAQ-based quantitative proteomic analyses of Landrace (LR) and Wuzhishan (WZS) pig longissimus dorsi muscle tissues during early embryonic development. RESULTS: The iTRAQ-based early embryonic longissimus dorsi muscle study of LR and WZS ranging from 21 to 42 days post coitus (dpc) identified a total of 4431 proteins from 17,214 peptides, which were matched with 36,4025 spectra at a false discovery rate of 5%. In both WZS and LR, the largest amount of differentially expressed proteins (DEPs) were found between 28 and 35 dpc. 252 breed-DEPs were selected by GO analysis, including 8 myofibrillar proteins. Only MYHCI/IIA mRNA were detected due to early embryonic stages, and significantly higher expression of them were found in WZS during these 4 stages. MYHCI was first found in WZS at 28 dpc and expressed in both breeds at later stages, while MYHCII protein was not detected until 35 dpc in both breeds. Thus, 33 myogenic breed-DEPs selected from last two stages were analyzed by STRING, which showed that some myofibrillar proteins (MYH1, TPM4, MYH10, etc.) and functional proteins (CSRP2, CASQ2, OTC, etc.), together with candidate myogenic proteins (H3F3A, HDGFRP2, etc.), probably participate in the regulatory network of myofiber formation. CONCLUSION: Our iTRAQ-based early embryonic longissimus dorsi muscle study of LR and WZS provides new data on the in vivo muscle development distinctions during early embryonic development, which contributes to the improved understanding in the regulation mechanism of early myogenesis in agricultural animals.

bFGF signaling-mediated reprogramming of porcine primordial germ cells.

Primordial germ cells (PGCs) have the ability to be reprogrammed into embryonic germ cells (EGCs) in vitro and are an alternative source of embryonic stem cells. Other than for the mouse, the systematic characterization of mammalian PGCs is still lacking, especially the process by which PGCs convert to pluripotency. This hampers the understanding of germ cell development and the derivation of authenticated EGCs from other species. We observed the morphological development of the genital ridge from Bama miniature pigs and found primary sexual differentiation in the E28 porcine embryo, coinciding with Blimp1 nuclear exclusion in PGCs. To explore molecular events involved in porcine PGC reprogramming, transcriptome data of porcine EGCs and fetal fibroblasts (FFs) were assembled and 1169 differentially expressed genes were used for Gene Ontology analysis. These genes were significantly enriched in cell-surface receptor-linked signal transduction, in agreement with the activation of LIF/Stat3 signaling and FGF signaling during the derivation of porcine EG-like cells. Using a growth-factor-defined culture system, we explored the effects of bFGF on the process and found that bFGF not only functioned at the very beginning of PGC dedifferentiation by impeding Blimp1 nuclear expression via a PI3K/AKT-dependent pathway but also maintained the viability of cultured PGCs thereafter. These results provide further insights into the development of germ cells from livestock and the mechanism of porcine PGC reprogramming.

Production of human apolipoprotein(a) transgenic NIBS miniature pigs by somatic cell nuclear transfer.

Most cases of ischemic heart disease and stroke occur as a result of atherosclerosis. The purpose of this study was to produce a new Nippon Institute for Biological Science (NIBS) miniature pig model by somatic cell nuclear transfer (SCNT) for studying atherosclerosis. The human apolipoprotein(a) (apo(a)) genes were transfected into kidney epithelial cells derived from a male and a female piglet. Male cells were used as donors initially, and 275 embryos were transferred to surrogates. Three offspring were delivered, and the production efficiency was 1.1% (3/275). Serial female cells were injected into 937 enucleated oocytes. Eight offspring were delivered (production efficiency: 0.9%) from surrogates. One male and 2 female transgenic miniature pigs matured well. Lipoprotein(a) was found in the male and one of the female transgenic animals. These results demonstrate successful production of human apo(a) transgenic NIBS miniature pigs by SCNT. Our goal is to establish a human apo(a) transgenic NIBS miniature pig colony for studying atherosclerosis.

Isolation and characterization of embryonic ameloblast lineage cells derived from tooth buds of fetal miniature swine.

Dental enamel formation, known as "amelogenesis," is initiated by cytodifferentiation of the ectodermally derived dental epithelium. Enamel cannot regenerate itself because once it is completely formed, ameloblasts are lost as the tooth erupts. Rodent teeth have been useful for studying the mechanisms of amelogenesis because ameloblast cell lines can be derived from the ever-growing incisors. However, higher mammals such as humans have no growing teeth, and cell lines derived from larger animals that are more similar to humans are required for higher fidelity studies. Here, we isolated embryonic enamel epithelium-derived epithelial cells from fetal swine. The explant culture of the developing deciduous molars that had been removed from the dental papilla-derived mesenchymal tissue and cells inside the tooth buds provided the epithelial cell population for the primary culture. To isolate the cell population, we performed a unique cell isolation technique called cell fishing. The isolated cells showed clear embryonic-stage ameloblast characteristics with appropriate gene/protein expressions of enamel matrix and proteinases, abundant glycogen pools, and secretory granular materials. They could be continuously subcultured several times and are presently being maintained. This cell population will facilitate the establishment of a stable cell line and allow us to characterize the definitive phenotype and functional behavior of porcine ameloblasts, which, in turn, promises to yield useful and practical findings that are more relevant than those provided by rodent studies. Finally, analysis of in vitro enamel formation will be important for engineering "bio-enamel" as a new dental therapy to restore enamel defects.

Nucleus transfer efficiency of ear fibroblast cells isolated from Bama miniature pigs at various ages.

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

The meganuclease I-SceI containing nuclear localization signal (NLS-I-SceI) efficiently mediated mammalian germline transgenesis via embryo cytoplasmic microinjection.

The meganuclease I-SceI has been effectively used to facilitate transgenesis in fish eggs for nearly a decade. I-SceI-mediated transgenesis is simply via embryo cytoplasmic microinjection and only involves plasmid vectors containing I-SceI recognition sequences, therefore regarding the transgenesis process and application of resulted transgenic organisms, I-SceI-mediated transgenesis is of minimal bio-safety concerns. However, currently no transgenic mammals derived from I-SceI-mediated transgenesis have been reported. In this work, we found that the native I-SceI molecule was not capable of facilitating transgenesis in mammalian embryos via cytoplasmic microinjection as it did in fish eggs. In contrast, the I-SceI molecule containing mammalian nuclear localization signal (NLS-I-SceI) was shown to be capable of transferring DNA fragments from cytoplasm into nuclear in porcine embryos, and cytoplasmic microinjection with NLS-I-SceI mRNA and circular I-SceI recognition sequence-containing transgene plasmids resulted in transgene expression in both mouse and porcine embryos. Besides, transfer of the cytoplasmically microinjected mouse and porcine embryos into synchronized recipient females both efficiently resulted in transgenic founders with germline transmission competence. These results provided a novel method to facilitate mammalian transgenesis using I-SceI, and using the NLS-I-SceI molecule, a simple, efficient and species-neutral transgenesis technology based on embryo cytoplasmic microinjection with minimal bio-safety concerns can be established for mammalian species. As far as we know, this is the first report for transgenic mammals derived from I-SceI-mediated transgenesis via embryo cytoplasmic microinjection.

Construction of a cDNA library for miniature pig mandibular deciduous molars.

BACKGROUND: The miniature pig provides an excellent experimental model for tooth morphogenesis because its diphyodont and heterodont dentition resembles that of humans. However, little information is available on the process of tooth development or the exact molecular mechanisms controlling tooth development in miniature pigs or humans. Thus, the analysis of gene expression related to each stage of tooth development is very important. RESULTS: In our study, after serial sections were made, the development of the crown of the miniature pigs' mandibular deciduous molar could be divided into five main phases: dental lamina stage (E33-E35), bud stage (E35-E40), cap stage (E40-E50), early bell stage (E50-E60), and late bell stage (E60-E65). Total RNA was isolated from the tooth germ of miniature pig embryos at E35, E45, E50, and E60, and a cDNA library was constructed. Then, we identified cDNA sequences on a large scale screen for cDNA profiles in the developing mandibular deciduous molars (E35, E45, E50, and E60) of miniature pigs using Illumina Solexa deep sequencing. Microarray assay was used to detect the expression of genes. Lastly, through Unigene sequence analysis and cDNA expression pattern analysis at E45 and E60, we found that 12 up-regulated and 15 down-regulated genes during the four periods are highly conserved genes homologous with known Homo sapiens genes. Furthermore, there were 6 down-regulated and 2 up-regulated genes in the miniature pig that were highly homologous to Homo sapiens genes compared with those in the mouse. CONCLUSION: Our results not only identify the specific transcriptome and cDNA profile in developing mandibular deciduous molars of the miniature pig, but also provide useful information for investigating the molecular mechanism of tooth development in the miniature pig.

Production of viable cloned miniature pigs by aggregation of handmade cloned embryos at the 4-cell stage.

The aim of the present study was to improve the quality of handmade cloned porcine embryos by multiple embryo aggregations. Embryos derived from aggregation of three cloned embryos (3×) had a better blastocyst rate than cloned control (1×) embryos (73.6% vs 35.1%, respectively; P<0.05), but did not differ from those produced by aggregation of two cloned embryos (2×; 63.0%). Total cell numbers differed among treatments (P<0.05), with the greatest cell numbers (126) in the 3× group and the lowest (55) in the control group. The ratio of inner cell mass:total cell number was comparable in the 2× and 3× groups (25.1% vs 26.1%, respectively) and was significantly better than that in the control group (15.3%). The proportion of apoptotic cells in 2× and 3× groups was lower than that in the control group (2.7% and 2.2% vs 4.7%, respectively; P<0.05). Expression of Oct4 and Cdx2 was higher, whereas that of Bax was lower (P<0.05), in the 3× compared with non-aggregate group. Seven piglets were born to two surrogate mothers after embryo transfer of 3× aggregated blastocysts. In conclusion, aggregated embryos had greater total cell numbers and better pluripotency gene expression, with reduced expression of the pro-apoptosis gene Bax. Collectively, these improvement may be associated with the development of cloned embryos to term.

Development of transgenic minipigs with expression of antimorphic human cryptochrome 1.

Minipigs have become important biomedical models for human ailments due to similarities in organ anatomy, physiology, and circadian rhythms relative to humans. The homeostasis of circadian rhythms in both central and peripheral tissues is pivotal for numerous biological processes. Hence, biological rhythm disorders may contribute to the onset of cancers and metabolic disorders including obesity and type II diabetes, amongst others. A tight regulation of circadian clock effectors ensures a rhythmic expression profile of output genes which, depending on cell type, constitute about 3-20% of the transcribed mammalian genome. Central to this system is the negative regulator protein Cryptochrome 1 (CRY1) of which the dysfunction or absence has been linked to the pathogenesis of rhythm disorders. In this study, we generated transgenic Bama-minipigs featuring expression of the Cys414-Ala antimorphic human Cryptochrome 1 mutant (hCRY1(AP)). Using transgenic donor fibroblasts as nuclear donors, the method of handmade cloning (HMC) was used to produce reconstructed embryos, subsequently transferred to surrogate sows. A total of 23 viable piglets were delivered. All were transgenic and seemingly healthy. However, two pigs with high transgene expression succumbed during the first two months. Molecular analyzes in epidermal fibroblasts demonstrated disturbances to the expression profile of core circadian clock genes and elevated expression of the proinflammatory cytokines IL-6 and TNF-α, known to be risk factors in cancer and metabolic disorders.

DNA methylation profiles provide a viable index for porcine pluripotent stem cells.

Porcine induced pluripotent stem cells (iPSCs) provide useful information for translational research. The quality of iPSCs can be assessed by their ability to differentiate into various cell types after chimera formation. However, analysis of chimera formation in pigs is a labor-intensive and costly process, necessitating a simple evaluation method for porcine iPSCs. Our previous study identified mouse embryonic stem cell (ESC)-specific hypomethylated loci (EShypo-T-DMRs), and, in this study, 36 genes selected from these were used to evaluate porcine iPSC lines. Based on the methylation profiles of the 36 genes, the iPSC line, Porco Rosso-4, was found closest to mouse pluripotent stem cells among 5 porcine iPSCs. Moreover, Porco Rosso-4 more efficiently contributed to the inner cell mass (ICM) of blastocysts than the iPSC line showing the lowest reprogramming of the 36 genes (Porco Rosso-622-14), indicating that the DNA methylation profile correlates with efficiency of ICM contribution. Furthermore, factors known to enhance iPSC quality (serum-free medium with PD0325901 and CHIR99021) improved the methylation status at the 36 genes. Thus, the DNA methylation profile of these 36 genes is a viable index for evaluation of porcine iPSCs.

Improvement of cloning efficiency in minipigs using post-thawed donor cells treated with roscovitine.

Massachusetts General Hospital miniature pigs (MGH minipigs) have been established for organ transplantation studies across the homozygous major histocompatibility complex, but cloning efficiency of MGH minipigs is extremely low. This study was designed to increase the productivity of MGH minipigs by nuclear transfer of post-thaw donor cells after 1 h co-incubation with roscovitine. The MGH minipig cells were genetically modified with GT KO (alpha1,3-galactosyltransferase knock-out) and hCD46 KI (human CD46 knock-in) and used as donor cells. The GT KO/hCD46 KI donor cells were cultured for either 3 days (control group) or 1 h after thawing with 15 µM roscovitine (experimental group) prior to the nuclear transfer. The relative percentage of the transgenic donor cells that entered into G0/G1 was 93.7 % (±2.54). This was different from the donor cells cultured for 1 h with the roscovitine-treated group (84.6 % ±4.6) (P < 0.05) and without roscovitine (78.6 % ±5.5) (P < 0.01), respectively. The pregnancy rate and delivery rate in the roscovitine group (8/12 and 6/8, respectively) were significantly higher (P < 0.01) than those in the control group (6/19 and 3/6, respectively). In the experimental group, 12 GT KO/hCD46 KI transgenic minipigs were successfully generated, and five minipigs among them survived for more than 6 months so far. The recipient-based individual cloning efficiency ranged from 0.74 to 2.54 %. In conclusion, gene-modified donor cells can be used for cloning of MGH minipigs if the cells are post-thawed and treated with roscovitine for 1 h prior to nuclear transfer.

Comparison of the efficiency of Banna miniature inbred pig somatic cell nuclear transfer among different donor cells.

Somatic cell nuclear transfer (SCNT) is an important method of breeding quality varieties, expanding groups, and preserving endangered species. However, the viability of SCNT embryos is poor, and the cloned rate of animal production is low in pig. This study aims to investigate the gene function and establish a disease model of Banna miniature inbred pig. SCNT with donor cells derived from fetal, newborn, and adult fibroblasts was performed, and the cloning efficiencies among the donor cells were compared. The results showed that the cleavage and blastocyst formation rates did not significantly differ between the reconstructed embryos derived from the fetal (74.3% and 27.4%) and newborn (76.4% and 21.8%) fibroblasts of the Banna miniature inbred pig (P>0.05). However, both fetal and newborn fibroblast groups showed significantly higher rates than the adult fibroblast group (61.9% and 13.0%; P<0.05). The pregnancy rates of the recipients in the fetal and newborn fibroblast groups (60% and 80%, respectively) were higher than those in the adult fibroblast group. Eight, three, and one cloned piglet were obtained from reconstructed embryos of the fetal, newborn, and adult fibroblasts, respectively. Microsatellite analyses results indicated that the genotypes of all cloning piglets were identical to their donor cells and that the genetic homozygosity of the Banna miniature inbred pig was higher than those of the recipients. Therefore, the offspring was successfully cloned using the fetal, newborn, and adult fibroblasts of Banna miniature inbred pig as donor cells.

Teratology studies in the minipig.

The minipig is a suitable species for regulatory teratology testing and may be regarded as an alternative to the rabbit, dog, and primate. The first successful regulatory teratology studies in the minipig were performed in the 1990s. It became clear that minipigs have several benefits over the other non-rodents, as they are purpose-bred for laboratory use, they are sexually mature at approximately 5 months of age, and they produce multiple offspring. The minipig has subsequently gained regulatory acceptance in the teratology testing of new drugs.