Genetic Modification of Pigs: Expanding their Utility as Biomedical Models

Development of transgenic animals has become technically more feasible over the past several decades creating opportunities for the genetic modification of numerous species. This dramatic expansion in the number of genetic modifications in pigs, in particular, is due to the advancement of molecular techniques that facilitate genetic modification as well as their agricultural importance and physiological characteristics, which make them an ideal model to conduct translational biomedical research. Genetic modification of pigs can address both basic and applied research questions with implications for agriculture and medicine

Long-Term Quercetin Dietary Enrichment Partially Protects Dystrophic Skeletal Muscle

Duchenne muscular dystrophy (DMD) results from a genetic lesion in the dystrophin gene and leads to progressive muscle damage. PGC-1α pathway activation improves muscle function and decreases histopathological injury. We hypothesized that mild disease found in the limb muscles of mdx mice may be responsive to quercetin-mediated protection of dystrophic muscle via PGC-1α pathway activation. To test this hypothesis muscle function was measured in the soleus and EDL from 14 month old C57, mdx, and mdx mice treated with quercetin (mdxQ; 0.2% dietary enrichment) for 12 months. Quercetin reversed 50% of disease-related losses in specific tension and partially preserved fatigue resistance in the soleus. Specific tension and resistance to contraction-induced injury in the EDL were not protected by quercetin. Given some functional gain in the soleus it was probed with histological and biochemical approaches, however, in dystrophic muscle histopathological outcomes were not improved by quercetin and suppressed PGC-1α pathway activation was not increased. Similar to results in the diaphragm from these mice, these data suggest that the benefits conferred to dystrophic muscle following 12 months of quercetin enrichment were underwhelming. Spontaneous activity at the end of the treatment period was greater in mdxQ compared to mdx indicating that quercetin fed mice were more active in addition to engaging in more vigorous activity. Hence, modest preservation of muscle function (specific tension) and elevated spontaneous physical activity largely in the absence of tissue damage in mdxQ suggests dietary quercetin may mediate protection

Long-term wheel running compromises diaphragm function but improves cardiac and plantarflexor function in the mdx mouse

Dystrophin-deficient muscles suffer from free radical injury, mitochondrial dysfunction, apoptosis, and inflammation, among other pathologies, which contribute to muscle fiber injury and loss leading to wheel chair confinement and death in the patient. For some time it has been appreciated that endurance training has the potential to counter many of these contributing factors. Correspondingly, numerous investigations have shown improvements in limb muscle function following endurance training in mdx mice. However, the effect of long-term volitional wheel running on diaphragm and cardiac function is largely unknown. Our purpose was to determine the extent to which long-term endurance exercise affected dystrophic limb, diaphragm and cardiac function. Following one year of volitional wheel running diaphragm specific tension was reduced by 60% (

Rescue of dystrophic skeletal muscle by PGC-1α involves restored expression of dystrophin-associated protein complex components and satellite cell signaling

Duchenne muscular dystrophy is typically diagnosed in the preschool years because of locomotor defects, indicative of muscle damage. Thus, effective therapies must be able to rescue muscle from further decline. We have established that Peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α) gene transfer will prevent many aspects of dystrophic pathology, likely through up-regulation of utrophin and increased oxidative capacity, however, the extent to which it will rescue muscle with disease manifestations has not been determined. Our hypothesis is that gene transfer of PGC-1α into declining muscle will reduce muscle injury compared to control muscle. To test our hypothesis AAV6 driving expression of PGC-1α was injected into single hind limbs of 3 week old mdx mice while the contralateral limb was given a sham injection. At six weeks of age treated solei had 37% less muscle injury compared to sham-treated muscles (p\u3c0.05). Resistance to contraction induced injury was improved 10% (p\u3c0.05) likely driven by the 5-fold (p\u3c0.05) increase in utrophin protein expression and increase in dystrophin associated complex members. Treated muscles were more resistant to fatigue, which was likely caused by the corresponding increase in oxidative markers. PGC-1α over-expressing limbs also exhibited increased expression of genes related to muscle repair and autophagy. These data indicate that the PGC-1α pathway remains a good therapeutic target as it reduced muscle injury and improved function using a rescue paradigm. Further, these data also indicate that the beneficial effects of PGC-1α gene transfer are more complex than increased utrophin expression and oxidative gene expression

Effects of Pre-Sorting Prior to Loading on Transport Losses of the Market Weight Pigs during Loading and Unloading

The objective of this study was to determine the effects Therefore, the objective of this study was to determine the effects of pre-sorting on stress responses at the time of loading and unloading and how it impacts transport losses in the market weight pig. This study took place between December and March. Thirty three loads of mixed sex market weight pigs (n = 5802) from three conventional grow-finish sites were used in a randomized complete block design. Each site had two rooms with both treatment groups represented in each room. The large pen, pre-sorted (LPS) treatment had 292 pigs/pen (0.67m2*pig -1). LPS pigs were sorted from pen mates ~18 h prior to loading. The large pen, not pre-sorted treatment (LNPS) also had 292 pigs/pen (0.67m2*pig-1). LNPS pigs were sorted from pen mates at the time of marketing. Pigs were moved in groups of four to six using sort boards and electric prods, when necessary. Treatments were randomly assigned to a trailer deck (~0.41 m2*pig-1). Straight deck trailers were used and pigs were transported ~1 h to a commercial harvest facility. During loading and unloading, the number of pigs displaying open mouth breathing (OMB), skin discoloration (SD), and muscle tremors (MT) were recorded. At the plant, dead and non-ambulatory pigs were recorded during unloading, and total losses were defined as the sum of dead and nonambulatory pigs at the plant. Data was analyzed using Proc Glimmix of SAS. Statistical analysis could not be run on the incidence of deads on arrival (DOA) because there were too many zeros in the dataset. LPS had two DOA’s (0.07%) and there were no DOA’s in the LNPS treatment. LPS pigs had lower (P ≤ 0.0001) OMB and SD during loading compared to LNPS pigs. There were no (P \u3e 0.05) differences for MT or non-ambulatory at loading or for stress responses at unloading. No (P \u3e 0.05) differences between treatments for fatigued, injured, total non-ambulatory or total losses existed. In conclusion, pre-sorting market weight pigs had some effect on reduced OMB and SD on farm; however, there were no differences for OMB, SD and MT or transport losses at the plant

Short-term heat stress alters redox balance in porcine skeletal muscle

Heat stress contributes to higher morbidity and mortality in humans and animals and is an agricultural economic challenge because it reduces livestock productivity. Redox balance and associated mitochondrial responses appear to play a central role in heat stress‐induced skeletal muscle pathology. We have previously reported increased oxidative stress and mitochondrial content in oxidative muscle following 12 h of heat stress. The purposes of this investigation were to characterize heat stress‐induced oxidative stress and changes in mitochondrial content and biogenic signaling in oxidative skeletal muscle. Crossbred gilts were randomly assigned to either thermal neutral (21°C; n = 8, control group) or heat stress (37°C) conditions for 2 h (n = 8), 4 h (n = 8), or 6 h (n = 8). At the end, their respective environmental exposure, the red portion of the semitendinosus muscle (STR) was harvested. Heat stress increased concentration of malondialdehyde (MDA) following 2 and 4 h compared to thermal neutral and 6 h, which was similar to thermal neutral, and decreased linearly with time. Protein carbonyl content was not influenced by environment. Catalase activity was increased following 4 h of heat stress and superoxide dismutase activity was decreased following 6 h of heat stress compared to thermal neutral conditions. Heat stress‐mediated changes in antioxidant activity were independent of altered protein abundance or transcript expression. Mitochondrial content and mitochondrial biogenic signaling were similar between groups. These data demonstrate that heat stress caused a transient increase in oxidative stress that was countered by a compensatory change in catalase activity. These findings contribute to our growing understanding of the chronology of heat stress‐induced intracellular dysfunctions in skeletal muscle

Acute heat stress activated inflammatory signaling in porcine oxidative skeletal muscle

Despite well‐studied clinical manifestations, intracellular mechanisms of prolonged hyperthermic injury remain unclear, especially in skeletal muscle. Given muscle\u27s large potential to impact systemic inflammation and metabolism, the response of muscle cells to heat‐mediated injury warrants further investigation. We have previously reported increased activation of NF‐κB signaling and increased NF‐κB and AP‐1‐driven transcripts in oxidative skeletal muscle following 12 h of heat stress. The purpose of this investigation was to examine early heat stress‐induced inflammatory signaling in skeletal muscle. We hypothesized that heat stress would increase NF‐κB and AP‐1 signaling in oxidative skeletal muscle. To address this hypothesis, 32 gilts were randomly assigned to one of four treatment groups (n = 8/group): control (0 h: 21°C) or exposed to heat stress conditions (37°C) for 2 h (n = 8), 4 h (n = 8), or 6 h (n = 8). Immediately following environmental exposure pigs were euthanized and the red portion of the semitendinosus muscle (STR) was harvested. We found evidence of NF‐κB pathway activation as indicated by increased protein abundance of NF‐κB activator IKK‐α following 4 h and increased total NF‐κB protein abundance following 6 h of heat stress. Heat stress also stimulated AP‐1 signaling as AP‐1 protein abundance was increased in nuclear fractions following 4 h of heat stress. Interleukin‐6 protein abundance and activation of the JAK/STAT pathway were decreased in heat stressed muscle. These data indicate that heat stress activated inflammatory signaling in the porcine STR muscle via the AP‐1 pathway and early activation of the NF‐κB pathway

Twelve hours of heat stress induces inflammatory signaling in porcine skeletal muscle

Heat stress causes morbidity and mortality in humans and animals and threatens food security by limiting livestock productivity. Inflammatory signaling may contribute to heat stress-mediated skeletal muscle dysfunction. Previously we discovered increased circulating endotoxin and intramuscular oxidative stress and TNFα protein abundance but not inflammatory signaling following 24 and 72 hours of heat stress. Thus, the purpose of this investigation was to clarify the role of inflammatory signaling in heat stressed skeletal muscle. Crossbred gilts (n=8/group) were assigned to either thermal neutral (24° C), heat stress (37° C), or pair-fed thermal neutral (24° C) conditions for 12 hours. Following treatment, animals were euthanized and the semitendinosus red (STR) and white (STW) were recovered. Heat stress did not alter inflammatory signaling in STW. In STR, relative heat shock protein abundance was similar between groups as was nuclear content of HSF1. In whole homogenate, relative abundance of the NF-κB activator IKKα was increased by heat stress though abundance of NF-κB was similar between groups. Relative abundance of phosphorylated NF-κB was increased by heat stress in nuclear fractions. AP-1 signaling was similar between groups. While there were few differences in transcript expression between thermal neutral and heat stress, 80 and 56% of measured transcripts driven by NF-κB or AP-1, respectively, were increased by heat stress compared to pair-fed thermal neutral. Heat stress also caused a reduction in IL-6 transcript and relative protein abundance. These data demonstrate that short-term heat stress causes inflammatory signaling through NF-κB in oxidative, but not glycolytic, skeletal muscle

Activin IIB receptor blockade attenuates dystrophic pathology in a mouse model of duchenne muscular dystrophy

Modulation of transforming growth factor-β (TGF-β) signaling to promote muscle growth holds tremendous promise for the muscular dystrophies and other disorders involving the loss of functional muscle mass. Previous studies have focused on the TGF-β family member myostatin and demonstrated that inhibition of myostatin leads to muscle growth in normal and dystrophic mice. We describe a unique method of systemic inhibition of activin IIB receptor signaling via adeno-associated virus (AAV)-mediated gene transfer of a soluble form of the extracellular domain of the activin IIB receptor to the liver. Treatment of mdx mice with activin IIB receptor blockade led to increased skeletal muscle mass, increased force production in the extensor digitorum longus (EDL), and reduced serum creatine kinase. No effect on heart mass or function was observed. Our results indicate that activin IIB receptor blockade represents a novel and effective therapeutic strategy for the muscular dystrophies

Farm and pig factors affecting welfare during the marketing process

The objective of this paper is to review the scientific literature to identify on-farm factors that contribute to market weight pig transportation losses. Transportation of market weight pigs is an essential element to the multisite pork production model used in the United States. In 2011 alone, approximately 111 million market weight pigs were transported from the finishing site to the abattoir. For pigs, the marketing process can present a combination of potentially novel, physical, and/or unfamiliar experiences that can be stressful. If the pig cannot cope with these sequential and additive stressors, then an increased rate of transportation losses could occur with a detrimental effect on pork carcass value. Current yearly estimates for transport losses are 1 million pigs (1%). A variety of market weight pig and farm factors have been reported to detrimentally affect transportation losses. By understanding how pigs interact with their environment during marketing, researchers, producers, and personnel at the abattoir may begin to identify, prioritize, and attempt to minimize or eliminate these stressors. This process will ultimately decrease transportation losses, improve pork quality, and increase profitability