Induction of lung progenitor cell-like organoids by porcine pluripotent stem cells.

Organoids are in vitro 3D models that are generated using stem cells to study organ development and regeneration. Despite the extensive research on lung organoids, there is limited information on pig lung cell generation or development. Here, we identified five epithelial cell types along with their characteristic markers using scRNA-seq. Additionally, we found that NKX2.1 and FOXA2 acted as the crucial core transcription factors in porcine lung development. The presence of SOX9/SOX2 double-positive cells was identified as a key marker for lung progenitor cells. The Monocle algorithm was used to create a pseudo-temporal differentiation trajectory of epithelial cells, leading to the identification of signaling pathways related to porcine lung development. Moreover, we established the differentiation method from porcine pluripotent stem cells (pPSCs) to SOX17+ FOXA2+ definitive endoderm (DE) and NKX2.1+ FOXA2+ CDX2- anterior foregut endoderm (AFE). The AFE is further differentiated into lung organoids while closely monitoring the differentiation process. We showed that NKX2.1 overexpression facilitated the induction of lung organoids and supported subsequent lung differentiation and maturation. This model offers valuable insights into studying the interaction patterns between cells and the signaling pathways during the development of the porcine lung.

Sophoridine Counteracts Obesity via Src-Mediated Inhibition of VEGFR Expression and PI3K/AKT Phosphorylation.

Sophoridine (SRP) is a natural quinolizidine alkaloid found in many traditional Chinese herbs, though its effect on adipose tissue is unclear. We improved serum lipid levels by administering SRP by gavage in high-fat diet (HFD)-fed C57BL/6 mice. After 11 weeks, SRP supplementation significantly reduced body weight gain and improved glucose homeostasis, while reducing subcutaneous fat and liver weight. SRP also inhibited cell proliferation and differentiation of 3T3-L1 cells. Proteomics analysis revealed that SRP inhibits adipocyte differentiation by interacting with Src, thereby suppressing vascular endothelial growth factor receptor 2 (VEGFR2) expression and PI3K/AKT phosphorylation. This study provides an empirical basis for the treatment of obesity with small molecules.

miR-103-3p Regulates the Proliferation and Differentiation of C2C12 Myoblasts by Targeting BTG2.

Skeletal muscle, a vital and intricate organ, plays a pivotal role in maintaining overall body metabolism, facilitating movement, and supporting normal daily activities. An accumulating body of evidence suggests that microRNA (miRNA) holds a crucial role in orchestrating skeletal muscle growth. Therefore, the primary aim of this study was to investigate the influence of miR-103-3p on myogenesis. In our study, the overexpression of miR-103-3p was found to stimulate proliferation while suppressing differentiation in C2C12 myoblasts. Conversely, the inhibition of miR-103-3p expression yielded contrasting effects. Through bioinformatics analysis, potential binding sites of miR-103-3p with the 3'UTR region of BTG anti-proliferative factor 2 (BTG2) were predicted. Subsequently, dual luciferase assays conclusively demonstrated BTG2 as the direct target gene of miR-103-3p. Further investigation into the role of BTG2 in C2C12 myoblasts unveiled that its overexpression impeded proliferation and encouraged differentiation in these cells. Notably, co-transfection experiments showcased that the overexpression of BTG2 could counteract the effects induced by miR-103-3p. In summary, our findings elucidate that miR-103-3p promotes proliferation while inhibiting differentiation in C2C12 myoblasts by targeting BTG2.

Adipose-specific BMP and activin membrane-bound inhibitor (BAMBI) deletion promotes adipogenesis by accelerating ROS production.

With the improvement of people's living standards, the number of obese patients has also grown rapidly. It is reported that the level of oxidative stress in obese patients has significantly increased, mainly caused by the increase in reactive oxygen species (ROS) levels in adipose tissue. Studies have shown that the use of siRNA to interfere with bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) expression could promote adipocyte differentiation, and under hypoxic conditions, BAMBI could act as a regulator of HIF1α to regulate the polarity damage of epithelial cells. In view of these results, we speculated that BAMBI may regulate adipogenesis by regulating the level of ROS. In this study, we generated adipose-specific BAMBI knockout mice (BAMBI AKO) and found that compared with control mice, BAMBI AKO mice showed obesity when fed with high-fat diet, accompanied by insulin resistance, glucose intolerance, hypercholesterolemia, and increased inflammation in adipose tissue. Interestingly, adipose-specific deficiency of BAMBI could cause an increase in the expression level of Nox4, thereby promoting ROS production in cytoplasm and mitochondria and the DNA-binding activity of C/EBPß and ultimately promoting adipogenesis. Consistently, our findings indicated that BAMBI may be a reactive oxygen regulator to affect adipogenesis, thereby controlling obesity and metabolic syndrome.

Comparative analysis of porcine iPSCs derived from Sertoli cells and fibroblasts.

Porcine embryonic fibroblasts (PEFs) can be directly reprogrammed into porcine induced pluripotent stem cells (piPSCs). However, the reprogramming process is generally lengthy and inefficient. Here, we established a fast and efficient induction system of piPSCs from porcine Sertoli cells (SCs) via forced expression of pig Yamanaka factors. The alkaline phosphatase (AP)-positive colonies from SCs developed on Day 3 after lentivirus infection, and were expanded and then picked up on Day 7, whereas reprogramming process from PEFs did not show any colonies in the same period. The picked piPSCs strongly expressed pluripotent genes, had the differentiation capacity to three germ layers, and could be also induced into primordial germ cell-like cells. Screening for transcription factor combinations showed that POU class 5 homeobox 1 (OCT4) is the core factor for AP-positive colony formation, and two factors (OCT4 and c-MYC) could successfully reprogram SCs into piPSCs. We then compared the RNA-sequencing data of piPSCs derived from SCs and PEFs, and found that the most significant difference was the activation of Transforming Growth Factor ß signaling pathway. We also compared the RNA levels of SCs and PEFs, and found that SCs exhibited higher Wnt signaling activity and Bone Morphogenetic Protein 4 expression than PEFs, which might be correlated with higher cell proliferation rate and reprogramming efficiency. In summary, the data demonstrated that starting cell sources of piPSCs significantly affect reprogramming dynamics and SCs could serve as cell sources for efficient reprogramming.

Deciphering the miRNA transcriptome of Rongchang pig longissimus dorsi at weaning and slaughter time points.

MicroRNA (miRNA) is essential for the process of gene posttranscriptional regulation in skeletal muscle of many species, such as mice, cattle and so on. However, a little number of miRNAs have been reported in the muscle development of Chinese native pig breeds. In this study, the longissimus dorsi transcripts of Chinese native Rongchang pig at weaning and slaughter time points were analysed for miRNA-seq. The results showed that 19 novel and 186 known miRNAs involved in the Rongchang pig skeletal muscle development were identified. Based on these findings, we further confirmed that porcine miR-127, miR-299 and miR-432-5p were obviously down-expressed in adult pig (287 days of age), while miR-7134-3p and 664-5p were significantly up-expressed in weaning pig (35 days of age). In other words, these miRNAs could be the potential molecular markers and play vital roles in the muscle development process. Moreover, we found miR-127 could inhibit the proliferation and myogenesis of porcine satellite cells in longissimus dorsi muscle. Our findings will provide deep insight into miRNA function for pork quality research with Chinese indigenous pig breeds.

Boar sperm quality and oxidative status as affected by rosmarinic acid at 17 °C.

Peroxidation damage induces sublethal injury to boar sperm during preservation. Rosmarinic acid (RA) has already been verified to efficiently protect cells from oxidant-induced injury and to produce significant effect on cryopreservation of semen. Through our experiments, we aim at investigating whether RA has a positive effect on the preservation of pig semen at room temperature. The semen collected from sexually mature Large White boars were preserved at 17 °C in Beltsville thawing solution (BTS) supplied. The boar sperm were exposed to 0, 25, 50, 75, 100, 125 and 150 µM RA in vitro and the sperm functions were examined. The sperm motility, the acrosome and plasma membrane integrity, the catalase activity (CAT), the total antioxidative capacity (T-AOC) activity and the malondialdehyde content (MDA) were examined at 0, 1, 3 and 5 days. The BTS diluent containing RA improved the sperm quality during the process of liquid preservation compared with the control treatment. After 5 days of liquid preservation, the addition of RA at 100 µM produced an optimal effect on the survival time as well as on the maintenance of motility, acrosome and plasma membrane integrity; T-AOC activity; CAT activity; and the MDA content. Besides, our results in the reproductive experiments showed that the addition of RA at 100 µM to the BTS diluent increased the pregnancy rate. These results suggest that the proper concentration of RA in boar semen extenders possibly improves the artificial insemination efficiency by reducing the sperm damage and the subsequent dysfunction during liquid preservation in swine production systems.

MicroRNA-664-5p promotes myoblast proliferation and inhibits myoblast differentiation by targeting serum response factor and Wnt1.

MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression at the post-transcriptional level and are involved in the regulation of the formation, maintenance, and function of skeletal muscle. Using miRNA sequencing and bioinformatics analysis, we previously found that the miRNA miR-664-5p is significantly differentially expressed in longissimus dorsi muscles of Rongchang pigs. However, the molecular mechanism by which miR-664-5p regulates myogenesis remains unclear. In this study, using flow cytometry, 5-ethynyl-2'-deoxyuridine staining, and cell count and immunofluorescent assays, we found that cell-transfected miR-664-5p mimics greatly promoted proliferation of C2C12 mouse myoblasts by increasing the proportion of cells in the S- and G2-phases and up-regulating the expression of cell cycle genes. Moreover, miR-664-5p inhibited myoblast differentiation by down-regulating myogenic gene expression. In contrast, miR-664-5p inhibitor repressed myoblast proliferation and promoted myoblast differentiation. Mechanistically, using dual-luciferase reporter gene experiments, we demonstrated that miR-664-5p directly targets the 3'-UTR of serum response factor (SRF) and Wnt1 mRNAs. We also observed that miR-664-5p inhibits both mRNA and protein levels of SRF and Wnt1 during myoblast proliferation and myogenic differentiation, respectively. Furthermore, the activating effect of miR-664-5p on myoblast proliferation was attenuated by SRF overexpression, and miR-664-5p repressed myogenic differentiation by diminishing the accumulation of nuclear ß-catenin. Of note, miR-664-5p's inhibitory effect on myogenic differentiation was abrogated by treatment with Wnt1 protein, the key activator of the Wnt/ß-catenin signaling pathway. Collectively, our findings suggest that miR-664-5p controls SRF and canonical Wnt/ß-catenin signaling pathways in myogenesis.

MiR-127 attenuates adipogenesis by targeting MAPK4 and HOXC6 in porcine adipocytes.

MicroRNAs (miRNAs) have critical roles during adipogenesis; however, their precise functions are not completely understood. Porcine miRNA expression profiles show that miR-127 is dramatically downregulated with age in adipose tissue. We aimed to identify the precise functions and mechanisms of miR-127 in proliferation and adipogenesis. Preadipocytes were cultured under conditions to induce proliferation or differentiation and the effect of miR-127 overexpression on these processes, and the associated bioinformatically predicted target genes, were assessed using luciferase assays, quantitative real-time PCR, western blot analysis, and cell staining techniques. miR-127 increased proliferation by promoting cell cycling, whereas it suppressed differentiation, which was accompanied by reduced lipid accumulation. miR-127 targeted mitogen-activated protein kinase 4 and homeobox C6 (HOXC6) to activate preadipocyte proliferation. During differentiation, miR-127 targeted HOXC6 to attenuate adipogenesis. These findings identify miR-127 as an inhibitor of porcine adipogenesis, which may inform future strategies to reduce porcine fat deposition and treat human obesity.

Wnt3a regulates mitochondrial biogenesis through p38/CREB pathway.

Wnt3a is established as an important regulator of various developmental processes, especially in osteogenesis, adipogenesis and mitochondrial biogenesis. Numerous studies reported Wnt3a regulates osteogenesis and adipogenesis, but the mechanisms by which Wnt3a regulates mitochondrial biogenesis are not well understood. In this study, results suggested that Wnt3a stimulates mitochondrial biogenesis by increasing the expression of mitochondrial biogenesis genes and regulators, as well as mitochondrial copy number in adipocytes. As a key mediator of canonical Wnt/ß-catenin pathway, ß-catenin knockdown had no effect on basal or Wnt3a-mediated mitochondrial biogenesis in adipocytes, which suggested that Wnt3a-mediated mitochondrial biogenesis was independent of ß-catenin-dependent canonical Wnt/ß-catenin pathway. However, Wnt3a inhibited p38/CREB (p38 mitogen-activated protein kinase/cAMP response element-binding protein) signaling activation and p38 inhibitor impaired Wnt3a-stimulated mitochondrial biogenesis, indicating p38/CREB pathway could be involved in the regulation of Wnt3a-mediated mitochondrial biogenesis in adipocytes. In conclusion, our data showed that Wnt3a stimulates mitochondrial biogenesis in adipocytes, which is at least partially through activation of p38/CREB pathway.

BAMBI shuttling between cytosol and membrane is required for skeletal muscle development and regeneration.

Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) gene encodes a transmembrane protein and is involved in multiple physiological and pathological processes, such as inflammatory response, tumor development and progression, cell proliferation and differentiation. A previous study suggested that BAMBI may interact with the Wnt/ß-catenin signaling pathway via promoting ß-catenin nuclear translocation associated with C2C12 myogenic myoblast differentiation. However, its biological function in skeletal muscle still remains unknown and requires further characterization. The present work sought to investigate its biological function in skeletal muscle, especially the physiological roles of BAMBI during skeletal muscle growth and regeneration. Our current work suggests that BAMBI protein is highly expressed in skeletal muscle and is only detected in cytosolic fraction in the resting muscle. Moreover, BAMBI protein is co-localized in fast-twitch (glycolytic) fibers, but not in slow-twitch (oxidative) fibers. Comparing with the cytosolic trapping in resting muscle, BAMBI protein is enriched on cellular membrane during the muscle growth and regeneration, suggesting that BAMBI-mediated a significant signaling pathway may be an essential part of muscle growth and regeneration.

MicroRNA-214-3p Targeting Ctnnb1 Promotes 3T3-L1 Preadipocyte Differentiation by Interfering with the Wnt/ß-Catenin Signaling Pathway.

Differentiation from preadipocytes into mature adipocytes is a complex biological process in which miRNAs play an important role. Previous studies showed that miR-214-3p facilitates adipocyte differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) in vitro. The detailed function and molecular mechanism of miR-214-3p in adipocyte development is unclear. In this study, the 3T3-L1 cell line was used to analyze the function of miR-214-3p in vitro. Using 5-Ethynyl-2'-deoxyuridine (EdU) staining and the CCK-8 assay, we observed that transfection with the miR-214-3p agomir visibly promoted proliferation of 3T3-L1 preadipocytes by up-regulating the expression of cell cycle-related genes. Interestingly, overexpression of miR-214-3p promoted 3T3-L1 preadipocyte differentiation and up-regulated the expression of key genes for lipogenesis: PPARγ, FABP4, and Adiponectin. Conversely, inhibition of miR-214-3p repressed 3T3-L1 preadipocyte proliferation and differentiation, and down-regulated the expression of cell cycle-related genes and adipogenic markers. Furthermore, we proved that miR-214-3p regulates 3T3-L1 preadipocyte differentiation by directly targeting the 3'-untranslated regions (3'UTR) of Ctnnb1, which is an important transcriptional regulatory factor of the Wnt/ß-Catenin pathway. Taken together, the data indicate that miR-214-3p may positively regulate preadipocyte proliferation and enhance differentiation through the Wnt/ß-Catenin signaling pathway.

miR-423-5p inhibits myoblast proliferation and differentiation by targeting Sufu.

Myoblast proliferation and terminal differentiation are the key steps of myogenesis. MicroRNAs are a class of small noncoding RNAs that play important roles in gene expression regulation. They negatively regulate gene expression by causing messenger RNA translational repression or target messenger RNA degradation. Here, we found that microRNA-423-5p (miR-423-5p) is highly expressed in both slow and fast muscles. Our gain-of-function study indicated that miR-423-5p actually plays a negative role in regulating myoblast proliferation and differentiation. We also found that miR-423-5p is able to inhibit the expression of suppressor of fused homolog to inactivate the expression of the marker genes in myoblast proliferation and differentiation. Taken together, our findings indicated miR-423-5p as a potential inhibitor of myogenesis by targeting suppressor of fused homolog in myoblast, and it also contributes to a better understanding of the microRNAs-target gene regulatory network in different types of porcine muscle types and may benefit the practice of improving the meat quality in animal husbandry.

Adiponectin AS lncRNA inhibits adipogenesis by transferring from nucleus to cytoplasm and attenuating Adiponectin mRNA translation.

Adiponectin (AdipoQ) is an adipocyte-derived hormone with positive function on systemic glucose and lipid metabolism. Long noncoding RNA (lncRNA) is emerging as a vital regulator of adipogenesis. However, AdipoQ-related lncRNAs in lipid metabolism have not been explored. Here, AdipoQ antisense (AS) lncRNA was first identified, and we further found that it inhibited adipogenesis. The half-life of AdipoQ AS lncRNA was 10 h, whereas that of AdipoQ mRNA was 4 h. During adipogenic differentiation, AdipoQ AS lncRNA translocated from nucleus to cytoplasm. AdipoQ AS lncRNA and AdipoQ mRNA formed an RNA duplex. Moreover, AdipoQ AS lncRNA delivered via injection of adenovirus expressing AdipoQ AS lncRNA decreases white adipose tissue (WAT), brown adipose tissue (BAT) and liver triglycerides (TG) in mice consuming a high fat diet (HFD). Interestingly, the non-overlapping region of AdipoQ AS lncRNA improved serum glucose tolerance and insulin sensitivity in HFD mice, but not AdipoQ AS lncRNA. In conclusion, AdipoQ AS lncRNA transfer from nucleus to cytoplasm inhibits adipogenesis through formation of an AdipoQ AS lncRNA/AdipoQ mRNA duplex to suppress the translation of AdipoQ mRNA. Taken together, we suggest that AdipoQ AS lncRNA is a novel therapeutic target for obesity-related metabolic diseases.

An Additive Effect of Promoting Thermogenic Gene Expression in Mice Adipose-Derived Stromal Vascular Cells by Combination of Rosiglitazone and CL316,243.

It is well-documented that CL316,243 (a ß3 agonist) or rosiglitazone (a PPARγ agonist) can induce white adipocyte populations to brown-like adipocytes, thus increasing energy consumption and combating obesity. However, whether there is a combined effect remains unknown. In the present study, stromal vascular cells of inguinal white adipose tissue (iWAT-SVCs for short) from mice were cultured and induced into browning by CL316,243, rosiglitazone, or both. Results showed that a combination of CL316,243 and rosiglitazone significantly upregulated the expression of the core thermogenic gene Ucp1 as well as genes related with mitochondrial function (Cidea, Cox5b, Cox7a1, Cox8b, and Cycs), compared with the treatment of CL316,243 or rosiglitazone alone. Moreover, co-treatment with rosiglitazone could reverse the downregulation of Adiponectin resulting from CL316,243 stimuli alone. Taken together, a combination of rosiglitazone and CL316,243 can produce an additive effect of promoting thermogenic gene expression and an improvement of insulin sensitivity in mouse iWAT-SVCs.

miR-429 Inhibits Differentiation and Promotes Proliferation in Porcine Preadipocytes.

MicroRNAs (miRNAs) are crucial regulatory molecules for adipogenesis. They contribute to the controlling of proliferation and differentiation of preadipocytes. Previous studies revealed an important role of miR-429 in cell invasion, migration, and apoptosis. Our previous work has shown that the expression of miR-429 in subcutaneous fat can be observed in newly born (3-day-old) Rongchang piglets rather than their adult counterparts (180-day-old). This expression pattern suggests that miR-429 might be functionally related to postnatal adipogenesis. However, we currently lack a mechanistic understanding of miR-429 within the context of preadipocyte differentiation. In this study, we investigated the function of miR-429 in porcine subcutaneous and intramuscular preadipocyte proliferation and differentiation. In our porcine preadipocyte differentiation model, miR-429 expression decreased remarkably upon adipogenic induction. Overexpression of miR-429 notably down-regulated the expression of adipogenic marker genes: PPARγ, aP2, FAS and impaired the triglyceride accumulation, while the expression of lipolytic gene ATGL was not affected. In addition, we observed that miR-429 significantly promoted the proliferation of porcine preadipocytes. We also found that miR-429 could directly bind to the 3'-UTRs of KLF9 and p27, which have been well documented to promote preadipocyte differentiation and repress cell cycle progression. Taken together, our data support a novel role of miR-429 in regulating porcine preadipocyte differentiation and proliferation, and KLF9 and p27 are potent targets of miR-429 during these processes.

3,3'-Diindolylmethane increases bone mass by suppressing osteoclastic bone resorption in mice.

3,3'-Diindolylmethane (DIM), a major acid-condensation product or metabolite of indole-3-carbinol which is found in cruciferous vegetables, has been shown to have anticancer, anti-inflammatory, and multiple immune stimulating effects. However, its function in bone metabolism is poorly understood. This study evaluated the effect of DIM on bone mass in mice under physiological and pathological conditions. Eight-week-old female mice received injections of a vehicle or 0.1mg/g of DIM, twice a week for four weeks. We found that DIM treatment significantly increased bone mass as assessed by dual-energy X-ray absorptiometry (DEXA) and micro-computed tomography (µCT). Further, Bone histomorphometric analyses showed that this treatment significantly reduced bone resorption parameters, but did not increase bone formation parameters. Furthermore, we use ovariectomized (OVX)-induced osteoporotic mouse model, and explore function of DIM in skeletal pathological processes. Bone phenotype analyses revealed that the administration of DIM in this study effectively prevented OVX-induced bone loss resulting from increased bone resorption. Our results demonstrated that DIM increased bone mass by suppressing osteoclastic bone resorption in bone metabolism under both physiological and pathological conditions. Accordingly, DIM may be of value in the treatment and the possible prevention of bone diseases characterized by bone loss, such as postmenopausal osteoporosis.

Aryl hydrocarbon receptor catabolic activity in bone metabolism is osteoclast dependent in vivo.

Bone mass is regulated by various molecules including endogenous factors as well as exogenous factors, such as nutrients and pollutants. Aryl hydrocarbon receptor (AhR) is known as a dioxin receptor and is responsible for various pathological and physiological processes. However, the role of AhR in bone homeostasis remains elusive because the cell type specific direct function of AhR has never been explored in vivo. Here, we show the cell type specific function of AhR in vivo in bone homeostasis. Systemic AhR knockout (AhRKO) mice exhibit increased bone mass with decreased resorption and decreased formation. Meanwhile, osteoclast specific AhRKO (AhR(ΔOc/ΔOc)) mice have increased bone mass with reduced bone resorption, although the mice lacking AhR in osteoblasts have a normal bone phenotype. Even under pathological conditions, AhR(ΔOc/ΔOc) mice are resistant to sex hormone deficiency-induced bone loss resulting from increased bone resorption. Furthermore, 3-methylcholanthrene, an AhR agonist, induces low bone mass with increased bone resorption in control mice, but not in AhR(ΔOc/ΔOc) mice. Taken together, cell type specific in vivo evidence for AhR functions indicates that osteoclastic AhR plays a significant role in maintenance of bone homeostasis, suggesting that inhibition of AhR in osteoclasts can be beneficial in the treatment of osteoporosis.

[The impact of phenolic compounds on pig fat deposition].

The quantity and distribution of fat deposits are crucial factors that impact the quality of pork. Recent studies have indicated that the utilization of natural ingredients plays a significant role in decreasing subcutaneous and visceral fat deposits, as well as enhancing intramuscular fat. Moreover, natural products possess several advantages including being environmentally friendly, safe, free of additives, and leaving no residue. Phenolic compounds derived from fruits, vegetables and herbs constitute of vital component of these natural ingredients. This article examines the influence of phenolic compounds on pig fat deposition, aiming to provide guidance on the utilization of such compounds to regulate fat deposition and enhance pork quality.

[Porcine skeletal muscle development regulated by MicroRNA: a review].

The growth and development of skeletal muscle is an important factor affecting pork production and quality, which is elaborately regulated by many genetic and nutritional factors. MicroRNA (miRNA) is a non-coding RNA with a length of about 22 nt, which binds to the 3'UTR sequence of the mRNA of the target genes, and consequently regulates its post-transcriptional expression level. In recent years, a large number of studies have shown that miRNAs are involved in various life processes such as growth and development, reproduction, and diseases. The role of miRNAs in the regulation of porcine skeletal muscle development was reviewed, with the hope to provide a reference for the genetic improvement of pigs.